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PubMed1995

Antiviral activity of a phosphorothioate oligonucleotide complementary to human cytomegalovirus RNA when used in combination with antiviral nucleoside analogs.

ISIS 2922 is a phosphorothioate oligonucleotide with potent antiviral activity against human cytomegalovirus (HCMV) in cell culture assays. The ability of ISIS 2922 to inhibit replication of HCMV when used in combination with other antiviral agents approved for treatment of HCMV disease was investigated using a 96-well immunoassay. The antiviral activity of ISIS 2922 against HCMV was additive with that of ganciclovir (9-(1,3-dihydroxy-2-propoxymethylguanine); DHPG) or foscarnet (phosphonoformate). Compounds used clinically for the treatment of human immunodeficiency virus infection and likely to be co-administered with ISIS 2922 in the clinic were also evaluated for their ability to modulate the antiviral activity of ISIS 2922. 3'-Azido-3'-deoxythymidine (AZT) exhibited no antiviral activity against HCMV in the 96-well immunoassay, and did not significantly alter the antiviral activity of ISIS 2922. 2'-3'-Dideoxycytidine (ddC) was able to inhibit replication of HCMV at high doses, and this activity was additive with that of ISIS 2922. ISIS 2922 inhibited HIV replication in acute infection assays at relatively high concentrations as previously reported for non-complementary phosphorothioate oligonucleotides. When ISIS 2922 was used in combination with AZT in this assay, interactions were additive at most concentrations, although significant and reproducible synergy was observed at some concentration combinations.

Antiviral Agents↗
PubMed1995

Toxicity, biodistribution and radioprotective capacity of L-homocysteine thiolactone in CNS tissues and tumors in rodents: comparison with prior results with phosphorothioates.

L-Homocysteine thiolactone (L-HCTL) was evaluated for its potential as an intravenously-administered central nervous system (CNS) radioprotector in C3H mice and F344 rats. Toxicity assessments in the mouse yielded a LD50 of 297 mg/kg and in the rat 389 mg/kg. Biodistribution studies in tumor-bearing mice showed that brain specimens contained more label at 10 min than the tumors but less at 30 or 60 min. Brain uptake relative to the tumors, the brain/tumor ratio, ranged between 0.5 and 3.3. The cervical spinal cord of non-tumor-bearing rats was irradiated with 32 Gy 137Cs with or without prior treatment with L-HCTL following which the time to forelimb or hindlimb paralysis was measured to determine the relative protective factors (RPFs) for this radiation dose. For forelimb paralysis the RPF was 1.9 (+/- 1.0, SD) and for hindlimb it was 2.0 (+/- 1.1, SD). 36B-10 glioma cells irradiated in vitro with or without L-HCTL and assayed for colony forming capacity demonstrated a dose modifying factor (DMF) of only 1.15 (+/- 0.16, SE). Rats bearing intracerebral 36B-10 glioma received 137Cs irradiation with or without L-HCTL after which the tumors were similarly assayed in vitro. From this the glioma DMF was 1.2 (+/- 0.30, SE). Compared to prior results with phosphorothioates our data show that the toxicity of L-HCTL is roughly the same as WR2721, WR77913 and WR3689 and that it distributes at higher levels in the CNS after systemic administration. L-HCTL may well equal these phosphorothioates at protecting normal CNS tissue without requiring administration directly into the cerebrospinal fluid-containing spaces and it does not protect the 36B-10 glioma.

Amifostine↗
PubMed1985

Some aspects of the toxicology of trimethyl and triethyl phosphorothioates.

The pharmacokinetics (disposal curves) of trimethyl and triethyl phosphorothioates have been determined. The concentrations to which the lung has been exposed at the LD50 dose of different chemical structures have been compared with the dose administered to the animal; the variation of LD50 of different chemical structures is little reduced. The in vitro kinetics of the reaction of O,S,S-trimethyl phosphorodithioate or O,O,S-triethyl phosphorothioate with plasma cholinesterase and carboxylesterase and brain acetylcholinesterase have been determined. The relation between inhibition and circulating concentrations in vivo have been examined. Changes in Clara cells reported by others seem to be physiological rather than pathological. O,S,S-Trimethyl phosphorodithioate is metabolised by rat lung and liver slices and microsomes. From these studies and the effect of various pretreatments of the rats on toxicity to the lung, it is probable that the proximal toxin is produced in the lung by oxidative attack on the alkylthio moeity of the compounds.

Animals↗
PubMed1993

Phosphorothioates in pre-tRNAs can change the specificities of RNAses P or reduce the cleavage efficiencies.

Several phosphorothioate-modified E coli and yeast pre-tRNAs were synthesized. If this modification included the phosphodiester at the RNase P cleavage site, two different effects were observed. With some pre-tRNAs the RNase P cleavage efficiency was severely reduced, whereas with other pre-tRNAs a new reaction type for RNase P was observed. Unlike the previously studied base or ribose modifications, phosphorothioates resulted in aberrant cleavages at unmodified phosphodiesters. These new sites could be located in the 5'-flank or in the acceptor stem of the tRNA domain. Modified mutants of E coli pre-tRNA(Tyr) with different base pairs at the RNase P cleavage site were cleaved with reduced efficiencies, but no aberrant products were observed.

Base Composition↗
PubMedDate not supplied

Sodium hydrogen-S-(3-amino-2-hydroxypropyl) phosphorothioate (WR-77913): toxicity and bone marrow radioprotection.

Experiments have been carried out to compare the toxicity and radioprotective effect of sodium hydrogen-S-(3-amino-2-hydroxypropyl) phosphorothioate (WR-77913) with those of S-2-(3-aminopropylamino)ethyl phosphorothioic acid (WR-2721). The drugs were given intraperitoneally to 12 week-old female BALB/c mice 30 minutes before whole body irradiation. Lethality at 30 days was the endpoint used. The drug LD50/30 was 678 mg/kg for WR-2721 and 3574 mg/kg for WR-77913. The LD50/30 for WR-77913 combined with 500 mg/kg of WR-2721 was 3328 mg/kg. The LD50/30 for misonidazole was 380 mg/kg when given in combination with 500 mg/kg of WR-2721 and 801 mg/kg when combined with 2200 mg/kg of WR-77913. Protection of bone marrow by WR-77913 and WR-2721 was comparable at doses close to the maximum tolerable dose (MTD, drug dose lethal to 10% of the animals at 30 days), but WR-77913 gave better protection at 35% of the MTD. These characteristics of low toxicity, non-additive toxicity with WR-2721, less toxicity in combination with misonidazole and adequate bone marrow protection at 25% of the MTD, make WR-77913 a protector worthy of further investigation.

Amifostine↗
PubMed1984

An improved HPLC assay for S-2-(3-aminopropylamino)ethyl phosphorothioate (WR-2721) in plasma.

An HPLC assay is presented for the detection and quantitation of the radioprotective drug S-2-(3-aminopropylamino)ethyl phosphorothioate (WR-2721, ethiofos) present in plasma. Improved selectivity and a 40-fold increase in sensitivity have been demonstrated over the method previously reported by this laboratory. Using precolumn derivatization with fluorescamine and S-3-(4-aminobutylamino)propyl phosphorothioate (WR-80855, a homolog of WR-2721) as the internal standard, drug levels of 0.05 to 1000 micrograms/mL were determined with excellent precision (CV less than or equal to 5% over the concentration range). An isocratic mobile phase of acetonitrile/ethanol/water (16:7:77) modified with 0.01 M tetrabutylammonium phosphate eluted the drug and the internal standard from the C-18 reverse-phase column in 23 minutes and 26 minutes, respectively. Detector response was linear over the entire range. The assay uses 150 microL of plasma and requires a total chromatography time of about 50 minutes. The method was found suitable for pharmacokinetic studies in a preliminary experiment with a beagle dog in which no interferences due to plasma constituents or drug metabolites were observed.

Amifostine↗
PubMed1988

Phosphorothioate and normal oligodeoxyribonucleotides with 5'-linked acridine: characterization and preliminary kinetics of cellular uptake.

Certain phosphorothioate oligodeoxynucleotide (S-oligo) analogs, unlike their normal congeners, have been found to exhibit significant anti-HIV activity [Matsukura et al., Proc. Natl. Acad. Sci. USA 84 (1987) 7706-7710]. Here we report melting temperatures (Tm) of a series of S-oligos compared with those of the corresponding normal oligomers. The Tm's for AT base pairs of S-oligos are significantly depressed relative to normal oligos, while GC-containing S-oligos show much less Tm depression. The Tm's of S-dT oligomers with poly(rA) are reduced relative to the duplexes with normal dA oligomers. These results provide a rational basis for the S-d(CG) sequences as anti-message inhibitors of gene expression. We also describe an automated synthesis of 5'-acridine linked oligothymidylates using phosphoramidite-linked acridine. During this synthesis we noted the replacement of thiophenol for the 6-chloro substituent on acridine. We have measured the Tm's of the compounds with 3 and 5 methylene groups linked to normal and phosphorothioate dTn (with n = 3-40) on duplex formation with the equivalent dAn, and have found small increases of Tm for the 5-methylene-linked acridine derivative. We have monitored the uptake of these fluorescently labeled oligos into HL60 cells, and found that the shorter oligos are more rapidly taken up than the longer, and the normal oligos faster than the S-oligos. The temperature dependence of the cellular uptake suggests an energy-dependent process, and a possible membrane receptor for oligos. These results have significance for the potential use of such compounds as inhibitors of gene expression.

Acridines↗
PubMed1995

Antisense phosphorothioates as antivirals against human immunodeficiency virus (HIV) and hepatitis B virus (HBV).

For the past 10 years we have focused on the development of antisense treatments against viral infections such as human immunodeficiency virus (HIV) and hepatitis B virus (HBV). We have demonstrated that phosphorothioate oligomers have in vitro anti-HIV activities and in vivo anti-HBV activity. In vitro anti-HIV activities of phosphorothioate oligodeoxynucleotides (S-oligo) were classified into sequence non-specific (non-antisense) and sequence specific (antisense). We found that toxicity of S-oligo could be also classified into the same 2 categories. Since some of non-specific toxicity is dependent on experimental conditions, in each experiment, we should pay attention to minimize non-specific toxicity of the oligomers.

Animals↗
PubMed1994

Quantitative analysis of phosphorothioate oligonucleotides in biological fluids using direct injection fast anion-exchange chromatography and capillary gel electrophoresis.

The analysis of antisense phosphorothioate DNA (SODN) in human plasma via direct injection using anion-exchange high-performance liquid chromatography (AE-HPLC) is presented. The method relies on the ability to selectively extract phosphorothioate DNA from undigested serum, plasma and urine on anion-exchange resins. The automated HPLC method can analyze a sample every 5 min with a limit of detection of 50 ng/ml (ppb). The DNA was collected, desalted and analyzed by capillary gel electrophoresis. Due to the high resolving power of this technique, a qualitative assessment of enzymatic degradation of the antisense oligonucleotide can be made.

Chromatography, Ion Exchange↗
PubMed1994

Development and validation of a method for routine base composition analysis of phosphorothioate oligonucleotides.

A method for routine base composition determination of phosphorothioate oligonucleotides containing as many as 21 nucleobases has been developed and systematically evaluated in terms of factors contributing to assay precision and accuracy. Phosphorothioate internucleotide linkages were oxidized with a mixture of tetrahydrofuran-water-methylimidazole (16:4:1, v/v/v) which has been shown to be 97.3% effective. This step was followed by enzymolysis and HPLC quantitation of individual nucleobases. RSD for inter-day base composition analysis ranged from 1.1 to 1.3%, and inter-lot variation was 0.6-2.0%. Accuracy of the determined nucleobase ratio was independently confirmed through sequencing of the oxidized oligomer by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS).

Base Composition↗
PubMed1989

Phosphorothioates in molecular biology.

The observation that phosphorothioate analogues of the nucleoside triphosphates are substrates for DNA- and RNA-polymerases has proven a boon for the molecular biologist. As these phosphorothioate-containing polymers are stable to degradation by nucleases and the sulfur atom confers many favourable chemical properties, several applications in molecular biology have been developed, including new methods for site-directed mutagenesis and DNA sequencing.

DNA↗
PubMed2006

Effect of the extent of thiolation and introduction of phosphorothioate internucleotide linkages on the anti-HIV activity of Suligovir [(s4dU)35].

Suligovir is a 35-mer homo-oligonucleotide, containing exclusively 4-thio deoxyuridylate, proved to be a potent inhibitor of HIV entry. In this paper, we described the effect of extent of thiolation and the introduction of nuclease-resistant phosphorothioate linkages on the anti-HIV activity of Suligovir. We found that the decreased thiolated nucleotide content decreases the anti-HIV potency of the compound and the introduction of phosphorothioate linkages does not improve its antiviral activity.

Anti-HIV Agents↗
PubMed2005

Electrochemical detoxification of four phosphorothioate obsolete pesticides stocks.

The phosphorothioate pesticides are widely used for crop production and fruit tree treatment, but their disposal causes serious environmental problems. Four commercial phosphorothioate pesticides (Demeton-S-methyl, Metamidophos, Fenthion and Diazinon) were treated by an electrolysis system using Ti/Pt as anode and stainless steel 304 as cathode. A number of experiments were run in a laboratory scale pilot plant and the results are presented. For Fenthion the achieved reduction was over 60%, while for Demeton-S-methyl, Metamidophos and Diazinon was more than 50%. Diazinon had the lowest energy demand. The COD/BOD5 ratio was improved considerably after electrolysis for all four pesticides examined. As a conclusion, electrochemical oxidation could be used as a pretreatment method of the pesticides detoxification.

Electrochemistry↗
PubMed2005

Immunostimulatory oligodeoxynucleotides induce dolphin neutrophil NADPH-oxidase activation in a CpG-independent but phosphorothioate backbone-dependent manner.

Immunocytes, which include antigen-presenting cells, B cells, natural killer cells and neutrophils, can be stimulated directly or indirectly with bacterial DNA and synthetic oligodeoxynucleotides (ODNs) with different structures and sequences. In the present study, we investigated the effect of synthetic ODNs on the respiratory burst of dolphin neutrophils using a chemiluminescence assay. Phosphorothioate (PS)-ODNs dose-dependently induced the respiratory burst, while phosphodiester (PO)-ODNs did not, regardless of CpG-content. The PS-ODN-induced activity was completely abolished by the flavoprotein inhibitor diphenyleneiodonium, which indicates that the NADPH-oxidase is activated by PS-ODNs. These results reveal that PS-ODNs induce dolphin neutrophil NADPH-oxidase activation in a CpG motif-independent but phosphorothioate-dependent manner.

Animals↗
PubMed1992

Template. Phosphorothioate oligonucleotides duplexes as inhibitors of HIV-1 reverse transcriptase.

We have investigated the interaction between a number of 14 mers phosphorothioate oligonucleotides and HIV-1 reverse transcriptase. Two methods were used to measure the affinity of the analogs for the enzyme. In the first, the oligonucleotide or its duplex with Poly(rl) were used as inhibitors of the enzyme using Poly(rA).(dT)14 as template primer. In the second, the oligonucleotides or their duplexes were used to displace a fluorescent template primer complex of known affinity from its binding site on reverse transcriptase. The two methods gave the same relative order of affinity. Phosphorothioate oligodeoxyribonucleotides had a much higher affinity than oligo(dC)14 and it was increased on hybridization. Quantitatively similar results were obtained for S(dC)14 or its analog with bases in the alpha-configuration. Of the analogs tested, only S(dC)14 showed priming activity.

Antiviral Agents↗
PubMed1997

Scavenger receptor-mediated delivery of antisense mini-exon phosphorothioate oligonucleotide to Leishmania-infected macrophages. Selective and efficient elimination of the parasite.

Targeted delivery of a 17-mer antisense phosphorothioate oligodeoxyribonucleotide, complementary to the common 5'-end of every mRNA of the parasite cells, to the phagolysosomes of cultured murine macrophages infected with Leishmania mexicana amazonensis selectively and efficiently eliminated the parasite cells without causing any detectable harm to the host cells. The antisense mini-exon oligonucleotide (ASM) was encapsulated into liposomes coated with maleylated bovine serum albumin (MBSA), the artificial ligand for macrophage scavenger receptors. MBSA-coating of the liposomes allowed specific binding of the liposomes to the macrophages, their receptor-mediated uptake, and subsequent degradation of the liposomes inside macrophage phagolysosomes to release ASM. When incubated with Leishmania-infected macrophages, MBSA-liposome-encapsulated ASM (10 microM) was able to kill >90% of the parasites within 5 hr as compared with 20% killing within this time period by free ASM. Oligonucleotides with complementary nucleotide sequence or with the same base composition as ASM but scrambled sequence had no antileishmanial effect under the conditions of the assay. This study reflects the efficacy of scavenger-receptor-mediated delivery of antisense phosphorothioate oligos in killing intraphagolysosomal pathogens.

Animals↗
PubMed1998

Co-administration of polyanions with a phosphorothioate oligodeoxynucleotide (CGP 69846A): a role for the scavenger receptor in its in vivo disposition.

The effects of co-administering polyanions on the pharmacokinetics of a 20-mer phosphorothioate oligodeoxynucleotide (CGP 69846A), and the role of scavenger receptors in its in vivo disposition, have been investigated. Following i.v. administration, CGP 69846A was rapidly cleared from the plasma and distributed amongst high (e.g. kidney, liver, spleen), low (e.g. skeletal muscle) and negligible (e.g. brain) accumulating tissues. In addition it was shown that: 1) dextran sulphate co-administration has a dose-dependent effect on the disposition of CGP 69846A; 2) CGP 69846A undergoes renal filtration and renal accumulation largely results from tubular reabsorption; 3) cross-inhibition studies are consistent with CGP 69846A being recognized by scavenger receptors in vitro and in vivo; and 4) the scavenger receptor may be an important determinant for the in vivo disposition of CGP 69846A in mice. These studies contribute toward an increased understanding of the mechanism underlying the pharmacokinetic behaviour of phosphorothioate oligodeoxynucleotides.

Animals↗
PubMed2003

Dynamic NMR structures of [Rp]- and [Sp]-phosphorothioated DNA-RNA hybrids: is flexibility required for RNase H recognition?

Chemically modified DNA oligonucleotides have been crucial to the development of antisense therapeutics. High-resolution structural studies of pharmaceutically relevant derivatives have been limited to only a few molecules. We have used NMR to elucidate the structure in solution of two DNA-RNA hybrids with the sequence d(CCTATAATCC).r(GGAUUAUAGG). The two hybrids contain an unmodified RNA target strand, whereas the DNA strand contains one of two different stereoregular sugar-phosphate backbone linkages at each nucleotide: 1), [Rp]-phosphorothioate or 2), [Sp]-phosphorothioate. Homonuclear two-dimensional spectroscopy afforded nearly complete nonlabile proton assignments. Distance bounds, calculated from the nuclear Overhauser effect (NOE) crosspeak intensities via a complete relaxation matrix approach with the program MARDIGRAS, were used to restrain the structure of the two hybrids during simulations of molecular dynamics. Analysis of restrained molecular dynamics trajectories suggests that both hybrids are flexible, requiring the use of molecular dynamics with time-averaged restraints (MDtar) to generate ensembles of structures capable of satisfying the NMR data. In particular, the deoxyribose sugars of the DNA strand show strong evidence of repuckering. Furthermore, deoxyribose sugar repuckering is accompanied by increased flexibility of overall helical geometry. These observations, together with the analysis of the crystal structure of a hybrid duplex in complex with ribonuclease H (RNase H), suggested that this flexibility may be required for recognition by RNase H.

Base Sequence↗