In Vitro and In Vivo Anti-influenza A Virus Activity of Antisense Oligonucleotides

Abstract

We have demonstrated that antisense phosphorothioate oligonucleotides (S-ODNs) inhibit influenza virus A replication in MDCK cells. The liposomally encapsulated and the free antisense phosphorothioate oligonucleotides with four target sites (PB1, PB2, PA, and NP) were tested for their abilities to inhibit virus-induced cytopathogenic effects by a MTT assay using MDCK cells. The liposomally encapsulated S-ODN complementary to the sites of the PB2-AUG initiation codon showed highly inhibitory effects. Therefore, the antiviral effects of S-ODN-PB2-AUG and PA-AUG were examined in a mouse model of influenza virus A infection. PB2-AUG oligomer treated i.v. significantly prolonged the mean survival time in day (MDS) and increased the survival rates with does dependent manner.     

Protein kinase CK1 is a p53-threonine 18 kinase which requires prior phosphorylation of serine 15

Human telomerase might be associated with malignant tumor development and could be a highly selective target for antitumor drug design. Antisense phosphodiester (ODNs) and phosphorothioate (S-ODNs) oligonucleotides were investigated for their abilities to inhibit telomerase activity in the HeLa cell line. The ODNs and S-ODNs were designed to be complementary to nucleotides within the RNA active site of telomerase. As a transfection reagent, FuGENE6 was used to enhance the cellular uptake of oligonucleotides in cell cultures. The results showed that S-ODN-3 (19-mer) encapsulated with FuGENE6 clearly inhibited the telomerase activity in HeLa cells, and the inhibitory efficiency increased with an increase in the S-ODN-3. However, free S-ODN-3 showed no inhibitory activity. On the other hand, ODN-3 encapsulated with FuGENE6 had no detectable inhibitory activity. The encapsulated S-ODNs exhibited higher inhibitory activities than the free S-ODNs, and showed sequence specific inhibition. Thus, the activities of the S-ODNs were effectively enhanced by using the transfection reagent. The transfection reagent, FuGENE6, may thus be a potentially useful delivery vehicle for oligonucleotide-based therapeutics and transgenes, and is appropriate for use in vitro and in vivo.     

Specific inhibition of transforming growth factor-beta2 expression in human osteoblast cells by antisense phosphorothioate oligonucleotides.

To elucidate the role of endogenous transforming growth factor (TGF)-beta2 on human osteoblast cell, antisense phosphorothioate oligonucleotides (S-ODNs) complementary to regions in mRNA of TGF-beta2 were synthesized and examined their effects on TGF-beta2 production and cell proliferation in a human osteoblast cell line ROS 17/2. Antisense S-ODNs were designated for three different target regions in the mRNA of TGF-beta2. Among several antisense S-ODN analyzed, an oligonucleotide (AS-11) complementary to the translation initiation site of mRNA of TGF-beta2 demonstrated a selective and strong inhibitory effect on TGF-beta2 production in osteoblast cells. Other antisense S-ODNs which were designated for other regions in mRNA of TGF-beta2 and one- or three-base mismatched analogs of AS-11 showed little or much less antisense activities than AS-11. Therefore, the most effective target site in mRNA of TGF-beta2 is at the initiation codon region. The antisense effects of AS-11 were observed without reduction of levels of mRNA of TGF-beta2. Furthermore, the inhibition of TGF-beta2 expression by antisense S-ODN appeared to enhance cell proliferation, demonstrating the growth inhibitory effect of autocrine TGF-beta2 in osteoblast cells.     

In vitro concentration dependent transport of phenytoin and phenobarbital,but not ethosuximide,by human P-glycoprotein

AimsOne possible mechanism for epilepsy drug resistance is overexpression of P-glycoprotein in the blood–brain barrier, but whether (or which) antiepileptic drugs (AEDs) are transported by P-gp remains unclear. We evaluated AEDs as P-gp substrates using cell monolayers.Main methodsBi-directional transport assays and concentration equilibrium transport assays (CETAs) were performed for phenytoin (PHT), phenobarbital (PB), and ethosuximide (ESM) using wildtype Madin–Darby Canine Kidney II cell line MDCKII and porcine renal endothelial cell line LLC–PK1 cells and these cells transfected with human MDR1 cDNA to express P-gp.Key findingsWildtype cells demonstrated no efflux transport of PHT, PB, or ESM. In CETAs, both MDR1-transfected cell lines transported PHT from basolateral to apical when PHT loading concentrations were 5 or 10, but not 20 µg/ml. MDCK–MDR1 cells transported PB when initial concentrations were 10 or 20, but not 5 µg/ml. LLC–MDR1 did not transport PB. P-gp inhibitor verapamil blocked efflux transport. MDR1-transfected cells did not transport ESM at 5.6 or 56 µg/ml. Bi-directional transport assays demonstrated weak transport for PHT but not PB or ESM.SignificanceHuman P-gp transports PHT and PB, but not ESM, in a concentration dependent manner. CETA may be more sensitive than bi-directional assays to detect transport of drugs with high passive diffusion. Potential P-gp substrates should be tested at clinically relevant concentration ranges.     

Clinical Studies with Liposomal Doxorubicin

Abstract

To present data on a continuing phase I study of liposomal doxorubicin at this time may seem like a giant leap backwards. Liposomally encapsulated doxorubicin has been available for clinical use for some years. Phase I studies have been completed, and phase II studies have been conducted in patients with breast cancer (1,2). Two major obstacles to the commercial exploitation of drugs encapsulated in liposomes were evident. the first was to find a preparation that could be given safely to humans. the second was to develop a pharmaceutical preparation that could be commercially exploited by fulfilling the requirements for manufacture and sale as a drug. the first of these problems was solved a number of years ago, but only recently have we had preparations that fulfilled the second requirement. Two of these have entered clinical trials at Roswell Park Memorial Institute (RPMI). One of these, the liposomally encapsulated muramyl tripeptide derivative (MTP-PE), has been presented previously (3,4). Accordingly, in this paper we will focus on our studies of liposomally encapsulated doxorubicin.     

Liposome Coated Stents: A Method to Deliver Drugs to the Site of Action and Improve Stent Blood-Compatibility

Abstract

N-(phosphonacetyl)-L-aspartic acid (PALA) exhibits a considerable increase in its in vitro growth inhibitory potency when it is incorporated into liposomes. Encapsulation in negatively charged liposomes increases the growth inhibitory potency of PALA by up to 360 times. For CV1-P and L929 cells, encapsulation in liposomes prepared from neutral phospholipids does not increase the potency of PALA. the potency of encapsulated PALA is not dependent on the size of liposomes, being equally effective in all liposomes between 0.1 and 1 µm in diameter. the potency of PALA is greatest when it is incorporated into liposomes prepared from high phase transition temperature lipids. Exposure of cells to 7.5 mM ammonium chloride substantially inhibits the effects of both free and encapsulated PALA. the potency of free PALA is more sensitive to changes in exposure length than is the potency of encapsulated PALA. Consequently the difference in potency between free and encapsulated PALA is greatest when exposure length is short (1-2 hr). the considerable potency of encapsulated PALA makes this drug a possible treatment for tumors and ocular cicatricial diseases.     

Phosphorylation of the influenza A virus protein PB1-F2 by PKC is crucial for apoptosis promoting functions in monocytes

The 11^th influenza A virus (IAV) protein PB1-F2 is encoded by an alternative reading frame of the PB1 polymerase gene and found in the nucleus, cytosol and at the mitochondria of infected cells, the latter is consistent with experimental evidence for its pro-apoptotic function. Here, the function of PB1-F2 as a phosphoprotein was characterized. PB1-F2 derived from isolate IAV_PR8 and synthetic fragments thereof were phosphorylated in vitro by purified protein kinase C (PKC) and cellular extract. Constitutively active PKCα interacts with PB1-F2 in yeast two-hybrid assays. ³²P radiolabelling of transfected 293T cells revealed that phosphorylation of PB1-F2 is sensitive to inhibitors of PKC and could be increased by the PKC activator PMA. ESI-MS analysis and cellular expression of PB1-F2 mutants identified the positions Ser-35 as the major and the Thr-27 as an alternative PKC phosphorylation site. Infection of MDCK cells with recombinant IAV_PR8 lacking these PKC sites abrogated phosphorylation of PB1-F2 in vivo . Furthermore, infection of primary human monocytes with mutant viruses lacking these PB1-F2 phosphorylation sites resulted in impaired caspase 3 activation and reduced progeny virus titres, indicating that the integrity of the identified phosphorylation sites is crucial for a cell-specific function of PB1-F2 during virus replication.     

Magnesium deprivation inhibits a MEK–ERK cascade and cell proliferation in renal epithelial Madin-Darby canine kidney cells

AimsLoss of magnesium (Mg²⁺) inhibits cell proliferation and augments nephrotoxicant-induced renal injury, but the role of Mg²⁺ has not been clarified in detail. We examined the effect of extracellular Mg²⁺ deprivation on a MEK–ERK cascade and cell proliferation using a renal epithelial cell line, Madin-Darby canine kidney (MDCK) cells.Main methodsMDCK cells were cultured in Mg²⁺-containing or Mg²⁺-free media. A HA-tagged constitutively active (CA)-MEK1 and a dominant negative (DN)-MEK1 were transfected into MDCK cells. The level of protein was examined by Western blotting. The intracellular free Mg²⁺ concentration ([Mg²⁺]_i) was measured using a fluorescent dye, mag-fura 2. Cell proliferation was determined by WST-1 assay. Dead cells were identified by staining with annexin V-FITC and propidium iodide.Key findingsIn the presence of fetal calf serum (FCS), Mg²⁺ deprivation decreased phosphorylated-ERK1/2 (p-ERK1/2) levels and [Mg²⁺]_i. Re-addition of Mg²⁺ increased p-ERK1/2 levels, which were inhibited by U0126, a specific inhibitor of a MEK–ERK cascade. Glutathione-S-transferase pull-down and coimmunoprecipitation assays showed that CA-MEK1 and DN-MEK1 binds with ERK1/2 in the presence of Mg²⁺. In contrast, neither CA-MEK1 nor DN-MEK1 bound to ERK1/2 in the absence of Mg²⁺. These results indicate that the MEK–ERK cascade is regulated by [Mg²⁺]_i. Cell proliferation was increased by the treatment with FCS or the expression of CA-MEK1 in the presence of Mg²⁺, but was inhibited by Mg²⁺ deprivation. Mg²⁺ deprivation did not increase the number of dead cells.SignificanceMg²⁺ is involved in the regulation of the MEK–ERK cascade and cell proliferation in MDCK cells.     

Antisense oligonucleotides directed against the viral RNA polymerase gene enhance survival of mice infected with influenza A.

We have investigated the ability of antisense phosphorothioate oligonucleotides to enhance the survival of mice infected with influenza A virus. The oligonucleotides were complementary to sequences surrounding the translation initiation codons of the viral PB2 or PA genes (PB2-as or PA-as, respectively) of the influenza A virus RNA polymerases. Intravenous administration of PB2-as in a complex with a cationic liposome, Tfx-10, significantly prolonged the mean survival time in days and increased overall survival rates of mice infected with the influenza A virus. Liposomally encapsulated PB2-as inhibited viral growth in lung tissues and reduced pulmonary consolidations. Liposomally encapsulated PB2-as could be an effective therapeutic agent against influenza A virus.     

2'-O-methyl-modified phosphorothioate antisense oligonucleotides have reduced non-specific effects in vitro

Antisense oligodeoxynucleotides (ODNs) have biological activity in treating various forms of cancer. The antisense effects of two types of 20mer ODNs, phosphorothioate-modified ODNs (S-ODNs) and S-ODNs with 12 2′-O-methyl groups (Me-S-ODNs), targeted to sites 109 and 277 of bcl-2 mRNA, were compared. Both types were at least as effective as G3139 (Genta, Inc.) in reducing the level of Bcl-2 protein in T24 cells following a 4 h transfection at a dose of 0.1 µM. Circular dichroism spectra showed that both types formed A-form duplexes with the complementary RNA, and the melting temperatures were in the order of Me-S-ODN·RNA > normal DNA·RNA > S-ODN·RNA. In comparison with the S-ODN, the Me-S-ODN had reduced toxic growth inhibitory effects, was less prone to bind the DNA-binding domain A of human replication protein A, and was as resistant to serum nucleases. Neither type of oligomer induced apoptosis, according to a PARP-cleavage assay. Hybrids formed with Me-S-ODN sequences were less sensitive to RNase H degradation than those formed with S-ODN sequences. Despite this latter disadvantage, the addition of 2′-O-methyl groups to a phosphorothioate-modified ODN is advantageous because of increased stability of binding and reduced non-specific effects.     

Differential effects of mycophenolate mofetil and cyclosporine A on peripheral blood and cord blood natural killer cells activated with interleukin-2

Background aimsGraft-versus-host disease remains a major cause of death after hematopoietic stem cell transplantation. Cyclosporine (CsA) and mycophenolate mofetil (MMF) have been successfully used alone or in combination as prophylaxis for graft-versus-host disease. Although the effects of these drugs on T cells have been studied, little is known about the effects of both drugs on natural killer (NK) cells. We examined if the sensitivity of umbilical cord blood (CB) NK cells to MMF and/or CsA differs from their adult counterparts.MethodsAn approach that was based on flow cytometry and real-time polymerase chain reaction was used to assess the effects of MMF, CsA and the combination of both drugs on the viability, activation, proliferation and cytotoxicity of peripheral blood (PB) and CB NK cells after culture with interleukin-2.ResultsMMF alone or together with CsA induced cell death of CB NK cells but not of PB NK cells. MMF and CsA had differential effects on NK cell activation but significantly reduced proliferation of CB NK cells. MMF reduced perforin expression by PB NK cells, whereas CsA alone or together with MMF drastically decreased degranulation of CB and PB NK cells. However, neither affected cytokine secretion by PB and CB NK cells.ConclusionsThis study showed that CB NK cells were more sensitive to MMF and CsA than were PB NK cells. MMF and CsA had significant effects on NK cells that could jeopardize the beneficial effects of NK cells after hematopoietic stem cell transplantation.     

Determination of optimal sites of antisense oligonucleotide cleavage within TNFalpha mRNA

Antisense oligonucleotides provide a powerful tool in order to determine the consequences of the reduced expression of a selected target gene and may include target validation and therapeutic applications. Methods of predicting optimum antisense sites are not always effective. We have compared the efficacy of antisense oligonucleotides, which were selected in vitro using random combinatorial oligonucleotide libraries of differing length and complexity, upon putative target sites within TNFα mRNA. The relationship of specific target site accessibility and oligonucleotide efficacy with respect to these parameters proved to be complex. Modification of the length of the recognition sequence of the oligonucleotide library illustrated that independent target sites demonstrated a preference for antisense oligonucleotides of a defined and independent optimal length. The efficacy of antisense oligonucleotide sequences selected in vitro paralleled that observed in phorbol 12-myristate 13-acetate (PMA)-activated U937 cells. The application of methylphosphonate:phosphodiester chimaeric oligonucleotides to U937 cells reduced mRNA levels to up to 19.8% that of the untreated cell population. This approach provides a predictive means to profile any mRNA of known sequence with respect to the identification and optimisation of sites accessible to antisense oligonucleotide activity.     

Impact of delivery method on antiviral activity of phosphodiester,phosphorothioate, and phosphoryl guanidine oligonucleotides in MDCK cells infected with H5N1 bird flu virus

We have previously described nanocomposites containing conjugates or complexes of native oligodeoxyribonucleotides with poly-L-lysine and TiO₂ nanoparticles. We have shown that these nanocomposites efficiently suppressed influenza A virus reproduction in MDCK cells. Here, we have synthesized previously undescribed nanocomposites that consist of TiO₂ nanoparticles and polylysine conjugates with oligonucleotides that contain phosphoryl guanidine or phosphorothioate internucleotide groups. These nanocomposites have been shown to exhibit antiviral activity in MDCK cells infected with H5N1 influenza A virus. The nanocomposites containing phosphorothioate oligonucleotides inhibited virus replication ~130-fold. More potent inhibition, i.e., ~5000-fold or ~4600-fold, has been demonstrated by nanocomposites that contain phosphoryl guanidine or phosphodiester oligonucleotides, respectively. Free oligonucleotides have been nearly inactive. The antiviral activity of oligonucleotides of all three types, when delivered by Lipofectamine, has been significantly lower compared to the oligonucleotides delivered in the nanocomposites. In the former case, the phosphoryl guanidine oligonucleotide has appeared to be the most efficient; it has inhibited the virus replication by a factor of 400. The results make it possible to consider phosphoryl guanidine oligonucleotides, along with other oligonucleotide derivatives, as potential antiviral agents against H5N1 avian flu virus.     

Inhibition of human telomerase activity by antisense phosphorothioate oligonucleotides encapsulated with the transfection reagent, FuGENE6, in HeLa cells

Telomerase, a ribonucleoprotein, synthesizes telomeric repeats (TTAGGG) onto the ends of chromosomes to maintain the constant length of the telomere DNA, and its activity is detectable in approximately 85%-90% of primary human cancers. Thus, it is postulated that human telomerase might be associated with malignant tumor development and could be a highly selective target for antitumor drug design. Antisense phosphorothioate oligonucleotides (S-ODN) were investigated for their abilities to inhibit telomerase activity in the HeLa cell line. The S-ODN were designed to be complementary to nucleotides within the RNA active site of telomerase. As a transfection reagent, FuGENE6 (Boehringer Mannheim, Mannheim, Germany) was used to enhance the cellular uptake of the oligonucleotides in cell cultures. The S-ODN encapsulated with FuGENE6 clearly inhibited telomerase activity in HeLa cells and showed sequence-specific inhibition. The encapsulated S-ODN-3 with a 19-nucleotide, (nt) chain length had inhibitory effects similar to those of the 21-mer and 23-mer S-ODN sequences (S-ODN-4 and 5), but the 15-mer and 17-mer S-ODN sequences (S-ODN-1 and 2) failed to satisfactorily prevent telomerase activity. However, apoptotic HeLa cell death was not associated with telomerase inhibition. Furthermore, the encapsulated S-ODN did not appear to be cytotoxic in terms of the cell growth rate. The oligonucleotides encapsulated with the transfection reagent had enhanced cellular uptake, and cytoplasmic and nuclear localizations were observed. However, weak fluorescent signals were observed within the cytoplasms of HeLa cells treated with the free S-ODN-3. Thus, the activities of the S-ODN were effectively enhanced by using the transfection reagent. The transfection reagent, FuGENE6, may thus be a potentially useful delivery vehicle for oligonucleotide-based therapeutics and transgenes and is appropriate for use in vitro and in vivo.     

Dynamic Bis-Intercalation of a Homodimeric Thiazole Orange Dye in DNA: Evidence of Intercalator Creeping

Abstract

We have used one and two dimensional exchange ¹H NMR spectroscopy to characterize the dynamics of the binding of a homodimeric thiazole orange dye, 1,1′-(4,4,8,8-tetramethyl-4,8-diaza-undecamethylene)-bis-4-(3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)-quinolinium tetraiodide (TOTO), to double stranded DNA (dsDNA). The double stranded oligonucleotides used were d-(CGCTAGCG)₂ ( 1 ) and d(CGCTAGCTAGCG)₂ ( 2 ). TOTO binds preferentially to the (5′-CTAG-3′)₂ sites and forms mixtures of 1:1 and 1:2 dsDNA-TOTO complexes with 2 in ratios dependent on the relative amount of TOTO and the oligonucleotide in the sample. The dynamic exchange between preferential binding sites in the case of a 2:1 1 -TOTO mixture is an intermolecular exchange process between two binding sites on different oligonucleotides. In the case of the 1:1 2 -TOTO complex an intramolecular exchange process occur between two different binding sites on the same strand. Both processes were studied. The results demonstrate the ability of TOTO to migrate along a dsDNA strand in an intramolecular exchange process. The migration process (“creeping”) along the DNA strand is 6 times faster than the rate of intermolecular exchange between sites in two different oligonucleotides.     

Novel poricoic acids attenuate renal fibrosis through regulating redox signalling and aryl hydrocarbon receptor activation

BackgroundRenal fibrosis is the final manifestation of chronic kidney disease (CKD). Renal fibrosis is largely driven by oxidative stress and inflammation.PurposeThe aim of the current study was to identify novel poricoic acids from Poria cocos and investigated their antifibrotic effects and the underlying mechanism.MethodsIn this study, we identified six novel poricoic acids from Poria cocos and examined their antifibrotic effect using transforming growth factor-β1- (TGF-β1-) induced cultured human kidney proximal tubular epithelial cells (HK-2) and mice with unilateral ureteral obstruction (UUO).ResultsTreatment with six poricoic acids significantly inhibited TGF-β1-induced α-smooth muscle actin expression at both mRNA and protein levels in HK-2 cells. Three compounds with an intact carboxyl group at C-3 position showed a stronger inhibitory effect than that of other three compounds with esterified carboxyl group at the C-3 position. Mechanistically, poricoic acid ZM (PZM) and poricoic acid ZP (PZP) attenuate renal fibrosis through the modulation of redox signalling including the inhibition of proinflammatory nuclear factor kappa B (NF-κB) signalling and its target genes as well as the activation of antioxidative nuclear factor-erythroid-2-related factor 2 (Nrf2) signalling and its downstream target gene in both TGF-β1-induced HK-2 cells and UUO mice. PZM treatment and PZP treatment inhibit the upregulated aryl hydrocarbon receptor and they target the gene expression in UUO mice. Intriguingly, PZM treatment exhibits a stronger inhibitory effect than that of the PZP treatment. Structure–function relationship reveals that the carboxyl group at C-3 position is the most important bioactive function group in secolanostane tetracyclic triterpenoids against renal fibrosis.ConclusionsPZM and PZP attenuated renal fibrosis through the modulation of redox signalling and the aryl hydrocarbon receptor signalling pathway. Our findings will provide several promising leading compounds against renal fibrosis.