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PubMed1993

A rapid method for quantitation of oligodeoxynucleotide phosphorothioates in biological fluids and tissues.

A sensitive and simple method for the quantitation of oligonucleotide phosphorothioates in biological fluids and tissues is described. This method is based on the extraction of the oligonucleotide from the biological fluids and tissues and immobilization on a nylon membrane. The membrane-bound oligodeoxynucleotide phosphorothioate is then hybridized with labeled complementary oligonucleotide and exposed to X-ray film. The data on the film can be scanned and used to create a standard curve. The sensitivity of detection by the method described here will be useful to monitor the pharmacokinetics of oligonucleotides in bodily fluids and distribution in various tissues. The results indicate that the method is rapid and allows handling of a large number of samples at the same time.

Animals↗
PubMed1993

Cellular uptake of phosphorothioate oligodeoxynucleotides is negatively affected by cell density in a transformed rat tracheal epithelial cell line: implication for antisense approaches.

Antisense oligodeoxynucleotides have been used to specifically inhibit the expression of a variety of genes. In the process of testing phosphorothioate oligodeoxynucleotides (S-oligos) as specific inhibitors of transforming growth factor alpha synthesis, we observed that the efficiency of cellular uptake of S-oligos was not constant throughout the culture period but decreased as the cell densities increased. Our data suggest that the mechanism by which oligonucleotides enter cells is negatively affected by increased cell density. These results emphasize the importance of determining uptake kinetics before designing and interpreting antisense experiments using phosphorothioate oligonucleotides.

Animals↗
PubMed1994

Reduction of erbB2 gene product in mamma carcinoma cell lines by erbB2 mRNA-specific and tyrosine kinase consensus phosphorothioate antisense oligonucleotides.

A considerable reduction of up to 75% at the protein level of the erbB2 gene product was observed using phosphorothioate antisense oligonucleotides directed against specific sequences of the erbB2 mRNA. Antisense oligonucleotides used were the 14-mer (translation start region) and the 17-mer (3' translated region) all-phosphorothioate oligonucleotides (S-ODNs). Sense or random sequences were used as a control. The greatest reduction at the erbB2 protein level was obtained after a 24h incubation with a single dose of 2 microM antisense S-ODN. The erbB2-mRNA-specific and the tyrosine kinase consensus antisense S-ODNs were comparably effective in inhibiting erbB2 expression. Addition of the transfection reagent DOTAP diminished the efficiency of erbB2 protein reduction by antisense S-ODNs after an incubation period of 24h but was more effective after 48h compared to the application of antisense S-ODNs alone.

Base Sequence↗
PubMed1996

In vivo inhibition of hepatitis B virus gene expression by antisense phosphorothioate oligonucleotides.

While an important goal of treatment for hepatitis B is to prevent the development of hepatocellular carcinoma, there has been no effective therapy for it. Antisense oligodeoxynucleotide treatment could in principle inhibit hepatitis B virus gene expression and suppress tumor development. We used a mouse model for hepatocellular carcinoma, which is transgenic for the hepatitis B virus HBx gene, to study antisense phosphorothioate oligodeoxynucleotides. Among 2 series of sense and antisense oligodeoxynucleotides, only antisense sequences covering the initiation codon of the HBx gene effectively inhibited the expression of the HBx gene in the liver. Intraperitoneal injection of this antisense oligodeoxynucleotide thrice a week for 8 weeks resulted in the prevention of preneoplastic lesion development in the liver without inflammation in the liver or developmental disturbance of the mice. Antisense phosphorothioate oligodeoxynucleotides can inhibit the expression of a hepatitis B virus gene and may be a promising method for the prevention of hepatocellular carcinoma in hepatitis B virus infection.

Animals↗
PubMed1996

Inhibition of influenza virus RNA polymerase and nucleoprotein genes expression by unmodified, phosphorothioated, and liposomally encapsulated oligonucleotides.

We have demonstrated that antisense phosphodiester (ODNs) and phosphorothioate oligonucleotides (S-ODNs) inhibit CAT (chloramphenicol acetyltransferase) protein expression in the clone 76 cell line, which is a derivative of the murine C127 cell line. This cell line expresses the influenza virus RNA polymerase and nucleoprotein (NP) genes in response to treatment with dexamethasone. Phosphodiester, phosphorothioate, and liposomally encapsulated oligonucleotides with four target sites (PB1, PB2, PA, and NP) were synthesized and tested for inhibitory effects by a CAT-ELISA assay using the clone 76 cell line. The ODNs and S-ODNs complementary to the sites of the PB2-AUG and PA-AUG initiation codons showed highly inhibitory effects. On the other hand, the inhibitory effect of the S-ODNs targeted to PB1 was considerably decreased in comparison with the other three target sites. Liposome encapsulation afforded oligomer protection in serum-containing medium and substantially improved cellular accumulation. The liposomal encapsulated oligonucleotides exhibited higher inhibitory activity than the free oligonucleotides. The activities of the unmodified oligonucleotides are effectively enhanced by using the liposomal carrier.

Animals↗
PubMed1993

Administration of a phosphorothioate oligonucleotide antisense to murine endogenous retroviral MCF env causes immune effects in vivo in a sequence-specific manner.

Previous in vitro studies had suggested that a product of the env gene of murine MCF (polytropic)-related sequences plays a role in regulating lymphocyte activation. To determine whether such an effect occurs in vivo, we have studied mice injected with phosphorothioate oligonucleotides antisense to such sequences. Injection of mice with antisense to the initiation region of the env gene resulted in (i) increased spleen cell numbers, primarily due to an increase in splenic B cells, (ii) increased class II MHC expression on B cells, (iii) increased RNA and DNA synthesis, and (iv) increased numbers of Ig producing cells. These results obtained with the antisense to MCF-related env did not occur with two scrambled phosphorothioate oligonucleotides or with antisense oligonucleotides to the initiation region of the env gene of xenotropic or ecotropic retroviral sequences. These data suggest that products of certain endogenous retroviral sequences regulate lymphocyte activation in vivo.

Animals↗
PubMed2000

Effects of phosphorothioate modifications on precursor tRNA processing by eukaryotic RNase P enzymes.

The cleavage mechanism has been studied for nuclear RNase P from Saccharomyces cerevisiae, Homo sapiens sapiens and Dictyostelium discoideum, representing distantly related branches of the Eukarya. This was accomplished by using precursor tRNAs (ptRNAs) carrying a single Rp or Sp-phosphorothioate modification at the normal RNase P cleavage site (position -1/+1). All three eukaryotic RNase P enzymes cleaved the Sp-diastereomeric ptRNA exclusively one nucleotide upstream (position -2/-1) of the modified canonical cleavage site. Rp-diastereomeric ptRNA was cleaved with low efficiency at the modified -1/+1 site by human RNase P, at both the -2/-1 and -1/+1 site by yeast RNase P, and exclusively at the -2/-1 site by D. discoideum RNase P. The presence of Mn(2+ )and particularly Cd(2+) inhibited the activity of all three enzymes. Nevertheless, a Mn(2+ )rescue of cleavage at the modified -1/+1 site was observed with yeast RNase P and the Rp-diastereomeric ptRNA, consistent with direct metal ion coordination to the (pro)-Rp substituent during catalysis as observed for bacterial RNase P enzymes. In summary, our results have revealed common active-site constraints for eukaryotic and bacterial RNase P enzymes. In all cases, an Rp as well as an Sp-phosphorothioate modification at the RNase P cleavage site strongly interfered with the catalytic process, whereas substantial functional interference is essentially restricted to one of the two diastereomers in other RNA and protein-catalyzed hydrolysis reactions, such as those catalyzed by the Tetrahymena ribozyme and nuclease P1.

Animals↗
PubMed1995

Reaction between metabolically activated acetaminophen and phosphorothioate oligonucleotides.

Assessment of toxic or mutagenic risks associated with phosphorothioate oligonucleotides (PTO) is important. In vitro and in vivo data have shown that PTOs are nontoxic and nonmutagenic. However, these studies do not address interactions between PTOs and other compounds. The sulfur on PTOs may provide a novel reactive center on a DNA molecule for drug interactions. This study chose acetaminophen (ACAP) as a model drug because ACAP is oxidized to the reactive intermediate N-acetyl-p-benzoquinone imine (NAPQI), which reacts with sulfur-containing compounds. Reaction of dCTP(S) with NAPQI or activated ACAP formed a product distinct from the reactants. Analysis of the product by fast atom bombardment mass spectroscopy gave a molecular weight consistent with NAPQI bound to a sulfur. Higher-molecular-weight products were seen on a polyacrylamide gel electrophoresis after incubation of fluorescein-labeled PTO with NAPQI. These products were not seen after incubation of a phosphodiester oligonucleotide with NAPQI. 31P NMR analysis confirmed the existence of a heterogenous mixture of adducts between a PTO and NAPQI. Nonsequence-specific PTOs of various lengths were tested for their ability to reduce ACAP toxicity. Cell viability showed that larger PTOs provided greater protection. We evaluated the ability of NAPQI to cause mutations in the LacZ gene of pBluescript plasmid which contained phosphorothioate linkages at designed locations within the gene. In addition, the ability of ACAP to cause mutations in the HGPRT locus in cells grown in dATP(S)-containing medium was measured. No mutations were seen in either assay. Based upon these results, activated ACAP is reactive with PTOs in vitro, although this interaction is nontoxic and nonmutagenic.

Acetaminophen↗
PubMed1994

Phosphorothioate oligonucleotides inhibit the replication of lentiviruses and type D retroviruses, but not that of type C retroviruses.

Phosphorothioate analogs of oligodeoxynucleotides at a concentration of 2 microM protected Himalayan tahr cells from infection by caprine arthritis encephalitis virus (CAEV) and equine dermis cells from infection by equine infectious anemia virus (EIAV). The characteristics of this inhibition against these lentiviruses are similar to those previously described for the inhibition of HIV-1 in ATH8 cells [17]. Thus, the 28-mer homo-oligomer of cytidine [S-(dC)28] was at least as effective as three anti-sense sequences targeted to the LTR, gag, and env regions of CAEV. The effectiveness of homo-oligomers of equal length was in the order C >> A > T, and a random 28-copolymer with a composition of 2C:1G was as effective as S-(dC)28. Shorter oligonucleotides were less effective (28 > 14 > 5 mers) for all base compositions tested. While replication of a simian type D retrovirus was inhibited by S-(dC)28, this compound did not inhibit the cytopathogenicity of two type C retroviruses, amphotropic murine leukemia virus (MuLV), and baboon endogenous virus, when they were tested in the same cell lines used to support the replication of lentiviruses. Southern blot analysis of the high molecular weight DNA of drug-treated CAEV-infected cells showed that S-(dC)28 was acting at or before the reverse transcription step. Our present data and the earlier finding that S-(dC)28 is a potent in vitro inhibitor of the MuLV reverse transcriptase [15] suggest that S-(dC)28 is acting very early in the replication cycle of these lentiviruses. Since MuLV reverse transcriptase is inhibited in vitro, but its replication is not blocked in permissive cells, our data suggest that the phosphorothioate oligonucleotides are preventing virus attachment.

Animals↗
PubMed1991

Disulfide-linked oligonucleotide phosphorothioates: novel analogues of nucleic acids.

The synthesis of phosphorothioate analogues of oligonucleotides by the oxidation of deoxyadenosine 3',5'-bisphosphorothioate (3) was attempted. Cyclization of 3 is much more efficient than oligomerization under all the conditions investigated. However, a preformed oligonucleotide carrying a 5'-terminal phosphorothioate group undergoes efficient chain-extension when oxidized in the presence of 3.

Cyclic AMP↗
PubMed1987

Phosphorothioated binary complex of eukaryotic initiation factor eIF-2 and GDP inhibits protein synthesis in hemin-supplemented reticulocyte lysates.

The rabbit reticulocyte heme-regulated eIF-2 alpha kinase (HRI) utilizes adenosine-5'-0-(3-thiotriphosphate) (ATP-gamma-S) as a substrate for its autophosphorylation and activation, and for the phosphorylation of eIF-2. The phosphorothioated binary complex [eIF-2(alpha-[35S]P) . GDP], interacted with the reticulocyte reversing factor (RF) in in vitro assays, and inhibited the ability of RF to catalyze GDP exchange from (eIF-2 . [3H]GDP) complexes. The phosphorothioate residue in the binary complex was resistant to phosphatase action under protein synthesis conditions. eIF-2(alpha-[35S]P) . GDP inhibited protein synthesis in hemin-supplemented lysates with biphasic kinetics, but had no effect on protein synthesis in heme-deficient lysates. The data reported here indicate that phosphorylation of eIF-2 . GDP alone, through the ability of eIF-2(alpha-P) . GDP to bind and sequester RF, is sufficient to inhibit protein chain initiation in the reticulocyte lysate.

Adenosine Triphosphate↗
PubMed1991

Phosphorothioate analogues of oligodeoxyribonucleotide: synthesis and activity as inhibitors of replication of human immunodeficiency virus.

The modifications of oligodeoxyribonucleotides include replacement of the other chain either all-PS (S-ODNs), or end-capped with several PS (SO-ODNs) groups at both 3'- and 5'-ends. A general synthesis of phosphorothioate analogues of oligodeoxyribonucleotides is described using the new phosphite. In assays of HIV, oligomers (S-ODNs) with complete replacement of phosphodiesters with phosphorothioate groups were found to be very active. Finally of particular interest is S-ODNs-rev or tat (20mers) which possessed slightly higher anti-HIV activity than S-dC28 itself. By contrast, the unmodified oligomers (ODNs) as well as SO-ODNs did not have any inhibitory effect.

Antiviral Agents↗
PubMed1987

Induction of the hepatic microsomal cytochrome P-450 system by trialkyl phosphorothioates in rats.

Single i.p. doses of O,O,O-triethyl phosphorothioate [OOO-Et(S)], one of the suicide substrates for cytochrome P-450, caused a rapid increase of NADPH-cytochrome c reductase activity in rat liver microsomes. The increase was dose dependent but did not coincide with the recovery from the inhibition of drug-metabolizing activities. There was no change of Km value of the reductase in the induced state. The co-administration of cycloheximide repressed the stimulatory effect of OOO-Et(S), suggesting that a de novo synthesis of enzyme protein may be responsible for the increase in activity. Of four homologous tri-n-alkyl esters tested, the triethyl compound was the most effective at 24 and 48 hr after administration. Triethyl phosphate, the oxygen analog of OOO-Et(S), also caused an increase of the reductase activity, but carbon disulfide had no influence on this activity. Although O,O,S-triethyl phosphorodithioate [OOS-Et(S)] and its n-alkyl homologs also caused the inhibition of drug-metabolizing activities and the increase of the reductase activity, the recovery and the stimulation of enzyme activity were different from that of O,O,O-tri-n-aklyl phosphorothioates.

Animals↗
PubMed1995

Pharmacokinetics and tissue distribution in rats of an oligodeoxynucleotide phosphorothioate (GEM 91) developed as a therapeutic agent for human immunodeficiency virus type-1.

An antisense oligodeoxynucleotide phosphorothioate, namely gene expression modulator 91 (GEM 91), has been demonstrated to have significant anti-human immunodeficiency virus activity in various tissue culture models. The present study was undertaken to determine the pharmacokinetics and tissue distribution of GEM 91 in rats following i.v. bolus administration of 35S-radiolabeled GEM 91. Plasma disappearance curves for GEM 91-derived radioactivity could be described by the sum of two exponentials, with half-lives (mean +/- SEM) of 0.95 (+/- 0.07) and 47.57 (+/- 14.48) hr. Urinary excretion represented the major pathway of elimination of GEM 91, with 26.67 +/- 6.46% (mean +/- SD) of the administered dose excreted within 24 hr and 58.12 +/- 4.36% over 240 hr after GEM 91 administration. Fecal excretion was a minor pathway of elimination of GEM 91 with 1.4 +/- 0.62% (mean +/- SD) of the administered dose excreted over 24 hr and 8.54 +/- 0.64% over 240 hr. A wide tissue distribution of GEM 91 was observed. During the initial 30 min, the highest levels of tissue radioactivity were found in the kidney, liver, spleen, lungs, and heart. Radioactivity was retained over longer time periods in the kidneys, liver, heart, and intestine. Analyses of the extracted radioactivities from plasma, kidney, and liver by gel electrophoresis showed the presence of both intact GEM 91 and degradative products with smaller molecular weights. Radioactivity in urine was found to be degradative metabolites of GEM 91. Based on the experimental data, pharmacokinetic parameters for GEM 91 in each tissue and biological fluids were calculated using computer-based two-compartmental i.v. bolus or absorption models. This study is important not only in providing the basis for future studies of GEM 91 in humans, but also in understanding the pharmacology and toxicology of antisense oligodeoxynucleotide phosphorothioates, in general.

Animals↗
PubMed1994

Quantitative capillary gel electrophoresis assay of phosphorothioate oligonucleotides in pharmaceutical formulations.

Quantitative capillary gel electrophoresis (QCGE) has been developed for the accurate quantitation of a 21-mer phosphorothioate oligonucleotide, ISIS 2922, and its degradation products in an intravitreal formulation. The electrokinetic mode of injection employed by CGE necessitates formulation of the external reference standard in a sample matrix similar to that of the drug product and the use of an internal standard for improved accuracy and precision. The analytical method detailed in this paper has demonstrated the necessary accuracy, precision, linearity, range, selectivity and ruggedness for use in routine drug product analysis and stability monitoring of phosphorothioate oligonucleotides.

Capillary Action↗
PubMed1983

Transport and metabolism of N6- and C8-substituted analogs of adenosine 3',5'-cyclic monophosphate and adenosine 3'5'-cyclic phosphorothioate by the isolated perfused rat kidney.

The clearance and metabolism of N6-substituted (N6-dimethyl-), C8-substituted (8-bromo-, 8-p-chlorophenylthio- (PCPT-)), and exocyclic oxygen substituted phosphorothioate diastereomers (cAMPS(Sp)) and cAMPS (Rp)) of adenosine 3':5'-monophosphate (cyclic AMP, cAMP) has been studied in an isolated perfused rat kidney. The N6- and C8-substituted analogs of cyclic AMP (10-100 microM) were not cleared as rapidly as exogenous cyclic AMP and were metabolized: N6- and C8-substituted analogs of adenosine accumulated in perfusate and urine. All analogs exhibited net transtubular secretion, i.e. their urinary excretion rate greater than glomerular filtration rate. Probenecid (0.9 mM) included in the perfusate abolished transtubular secretion and inhibited the metabolism of PCPT-cyclic AMP, suggesting that cyclic AMP analogs, like cyclic AMP itself, penetrate the renal cell at the peritubular membrane by an organic acid transport system. The phosphorothioate diastereomers of cyclic AMP: cAMPS(Sp) and cAMPS(Rp) were cleared as rapidly from the perfusate as cyclic AMP, were extensively secreted (urinary excretion/ glomerular filtration greater than or equal to 10) and exhibited no metabolism. The latter analog would seem most suitable as an intracellular agonist for cyclic AMP-mediated phenomena in the rat kidney.

Animals↗
PubMed1987

A phosphorothionate isomer protects against the pneumotoxicity caused by O,O,S-trimethyl phosphorothioate.

O,O,S-Trimethyl phosphorothioate (OOS-TMP), an impurity in many organophosphorus insecticides, causes pneumotoxicity in rats at low doses (20 mg/kg) resulting in increases in bronchopulmonary lavage lactate dehydrogenase (LDH) activity and morphological alterations of bronchiolar epithelium. Coadministration of the nontoxic isomer, O,O,O-trimethyl phosphorothioate (OOO-TMP), at 1% of the toxicant dose, has been found to protect against the increase in LDH levels and morphological changes in bronchioles caused by OOS-TMP. Since OOO-TMP appears to require metabolic activation for pneumotoxicity, the effects of OOO-TMP on pulmonary and hepatic P-450 content and P-450-mediated monooxygenases were examined as a possible biochemical mechanism of antagonism. Oral treatment with OOO-TMP (0.5, 1.0, and 4.0 mg/kg) decreased pulmonary P-450 levels by 23 to 50% at 2 and 6 hr, while no changes were detected in hepatic P-450 levels. Lung microsomal 7-ethoxycoumarin O-deethylase (7-Ec) was inhibited by 71 to 100%, while liver 7-Ec was inhibited by 26 to 52%. p-Nitroanisole demethylase activity was decreased 22 to 47% following treatment with the two highest dose levels of OOO-TMP. These results further support the view that the lung is a target organ of delayed toxicity produced by OOS-TMP, and that the antagonistic effect of OOO-TMP is due to alterations in the metabolic activation processes of OOS-TMP in the lung and/or liver.

Animals↗
PubMed1990

Oligodeoxynucleoside phosphorothioate stability in subcellular extracts, culture media, sera and cerebrospinal fluid.

Degradation of a synthetic oligodeoxynucleoside phosphorothioate was studied in six systems used for antisense inhibition experiments. Oligodeoxynucleoside phosphorothioates were degraded very slowly at 37 degrees C in all of the systems studied. Measured half-lives of pentadecamers were 12 +/- 1 h in rabbit reticulocyte lysate, 7 +/- 1 h in HeLa cell postmitochondrial extract, 14 +/- 2 h in RPMI 1640 with 10% fetal bovine serum, 8 +/- 1 h in undiluted fetal bovine serum, 9 +/- 1 h in adult human serum, and 19 +/- 7 h in rat cerebrospinal fluid.

Animals↗