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PubMed1996

Dosimetry of radionuclide therapy using radiophosphonated antisense oligodeoxynucleotide phosphorothioates based on animal pharmacokinetic and tissue distribution data.

The aim of this study was to evaluate the therapeutic possibilities of radiolabeled antisense oligodeoxynucleotides. The internal radiation dose from known cellular and animal data was calculated, and the suitability of different phosphorus isotopes as labels for oligonucleotides was assessed. We calculated the pharmacokinetics and tissue distribution in vivo of short oligodeoxynucleotide phosphorothioates by using the data from two different radionuclides: phosphorus-33 (t1/2 = 24.4 days, maximum beta-energy = 250 ke V) and phosphorus-32 (t1/2 = 14.3 days, maximum beta-energy = 2270 ke V). The absorbed doses of 32P-labeled and 33P-labeled oligonucleotides were estimated using the published biodistribution data for several oligonucleotides in animal models for both tumor xenografts and AIDS. The local absorption of 33P was higher than that of 32P if the radius of the spherical distribution of activity was smaller than approximately 500 microns. In a mouse tumor xenograft model, an intravenously injected activity of 1 MBq achieved sufficient radiation doses in the tumor; 11 Gy for 32P and 1.5 Gy for 33P were obtained. In normal organs in the same model, the liver doses were 5.0 Gy (32P) and 0.7 Gy (33P), and the kidney doses were 14 Gy (32P) and 2.0 Gy (33P). We conclude that 33P may have more beneficial radiotherapeutic characteristics for oligonucleotides than 32P. This method could be applied on the macroscopic, cellular, and subcellular levels and help to design further experimental studies for the use of oligonucleotide radiotherapy and phosphorothioate probes.

Acquired Immunodeficiency Syndrome↗
PubMed1997

A nonradioisotope approach to study the in vivo metabolism of phosphorothioate oligonucleotides.

A 25-mer phosphorothioate oligodeoxynucleotide (GEM 91) complementary to the gag gene mRNA of HIV-1 virus was administered intravenously (i.v.) at a dose of 10 mg/kg/day for 8 weeks or 25 mg/kg single dose subcutaneously (SC) to adult Rhesus monkeys. No radioactive markers were used. A capillary gel electrophoresis (CGE) method with UV detection was used to determine the concentration of GEM 91 in plasma and the metabolite profile. The metabolite profile was virtually the same following a single dose of either 10 mg/kg i.v. or 25 mg/kg SC. A different metabolite profile was observed after 4 or 8 weeks of multiple i.v. doses of 10 mg/kg/day. The extract was subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) for positive identification. Mass spectrometry confirmed the major metabolic pathway in vivo to be via 3'-end exonuclease activity. The extract was then subjected to a hybridization-assisted ligation reaction in which only 5'-end intact metabolites were labeled. Analysis by CGE with laser-induced fluorescence (LIF) detection allowed each of these metabolites to be quantified with a limit of detection of 1 ppb (ng/ml). MALDI-TOFMS identified components digested from both ends of the DNA. This study demonstrates that the combination of quantitative CGE-LIF and MALDI-TOFMS yields a powerful and unique approach to study the metabolism of phosphorothioate oligonucleotides.

Animals↗
PubMed1999

Phosphorothioate oligodeoxynucleotides inhibit basic fibroblast growth factor-induced angiogenesis in vitro and in vivo.

Angiogenesis is regulated by heparin-binding growth factors, such as basic fibroblast growth factor (bFGF). We investigated the effects of phosphorothioate-mediated oligodeoxynucleotides (PS-ODN) on bFGF-induced angiogenesis. Because PS-ODN are polyanions, they can also bind many heparin-binding proteins. On a basement matrix using a Matrigel matrix, we observed <50% tube formation by human umbilical endothelial cells with 10 microM bFGF, vascular endothelial growth factor, or nuclear factor-kappaB (NF-kappaB) antisense and sense PS-ODN, while phosphodiester oligodeoxynucleotides (PO-ODNs) were not affected. The PS-ODN, but not the PO-ODN, inhibited the bFGF-induced rabbit corneal neovascularization. In albino rats, the NF-kappaB antisense PS-ODN showed a low rescue score for bFGF-dependent photoreceptor rescue because of their degradation by constant light exposure. However, antisense PS-ODN active against bFGF inhibited angiogenesis more strongly than did the antisense NF-kappaB PS-ODN. Because of the important role bFGF plays in angiogenesis, some PS-ODN may serve as potent antiangiogenic compounds that act through a combination of polyanionic phosphorothioate effects and a sequence-specific antisense mechanism.

Animals↗
PubMed1999

Comparison of binding of N3'-->P5' phosphoramidate and phosphorothioate oligonucleotides to cell surface proteins of cultured cells.

The binding of uniformly modified N3'-->P5' phosphoramidate and stereorandom and stereopure phosphorothioate oligonucleotides (ODN) to cell surface proteins was studied, using both a fibroblast and an epithelial cell line, to assess the effect of different analog backbone types and base composition on cell surface protein binding. Marked differences were observed, both quantitative and qualitative, in the proteins to which individual ODN bound. One phosphoramidate, antisense to the insulin-like growth factor-1 (IGF-1) receptor (IGF-1R), bound to different proteins than did either a 6-base mismatch phosphoramidate IGF-1R sequence or a sense N-ras sequence. The latter bound poorly to the fibroblast line and predominantly to a 46 kDa protein in the epithelial line, as did many of the other ODN. This binding was not so marked as that of the isosequential end-capped phosphodiester N-ras sequence, which bound to this protein in both cell lines. Stereopure and stereorandom phosphorothioates containing a G-quartet (shown in other studies to form high-order tetrad structures), antisense to c-myc, exhibited considerable nonspecific binding to many proteins, as did the isosequential phosphoramidate. In particular, this ODN sequence gave notable binding to high molecular weight proteins. In general, binding of the c-myc ODN to proteins of 28-30, 46, 67, and 70-90 kDa was found in this study.

3T3 Cells↗
PubMed2004

Distribution and excretion of a phosphorothioate oligonucleotide in rats with experimentally induced renal injury.

The effects of renal injury on the urinary excretion and tissue distribution of a 20-mer phosphorothioate oligonucleotide were investigated in male Sprague-Dawley rats. Renal injury was produced by treating the rats with either 5.0 mg/kg cisplatin or 2.5 mg/kg of a monoclonal antibody (mAb) directed toward Thy1.1. Controls received saline. Three days after cisplatin treatment or 2 days after anti- Thy1.1 treatment, the rats received 10 mg/kg ISIS 3521. Blood was collected at various times to assess the plasma concentrations of ISIS 3521, and rats were killed at various times from 6 to 48 hours after intravenous (i.v.) infusion of oligonucleotide to assess tissue concentrations by capillary gel electrophoresis (CGE). Cisplatin and anti-Thy1.1 antibody produced histologic and biochemical changes consistent with proximal tubular damage and glomerular damage, respectively. Urinary excretion of oligonucleotides was increased 2- to 4-fold of control; however, this amount accounted for only 1% to 2% of dose compared to 0.5% in controls. Proximal tubular damage reduced renal accumulations of ISIS 3521 and other oligonucleotide metabolites, but there were no obvious compensatory increases in concentrations in other organs except for a slight increase in spleen levels of total oligonucleotide. Glomerular damage was not associated with any change in oligonucleotide disposition. Immunohistochemical studies showed no evidence of alterations in the pattern of distribution within the injured kidney. The data suggest that acute renal dysfunction, either renal tubular or glomerular, does not markedly alter the urinary elimination and tissue deposition of a phosphorothioate oligonucleotide.

Animals↗
PubMed2006

G3139 and other CpG-containing immunostimulatory phosphorothioate oligodeoxynucleotides are potent suppressors of the growth of human tumor xenografts in nude mice.

Several phosphorothioate antisense oligodeoxynucleotides (ODN) are developed to target factors potentially involved in tumor growth and apoptosis suppression. Among them, the 18-mer G3139 (Oblimersen), which targets Bcl-2, is currently being tested in phase II and phase III clinical trials for various tumors in combination with chemotherapy. On the other hand, ODNs containing CpG dinucleotides (CpG-ODN) within specific-sequence contexts (CpG motifs) have been shown to activate rodent or primate immune cells via toll-like receptor 9 (TLR9) and have demonstrated remarkable T cell-dependent antitumor efficacy in a series of murine tumor models. However, immune cell activation by CpG-ODN is largely diminished upon C-5 methylation at CpG cytosine. As G3139 contains CpG motifs, we questioned whether the antitumor effects seen in human tumor xenografts might be abrogated by cytosine C-5 methylation of G3139, which retained the ability of G3139 to suppress Bcl-2 expression in tissue culture, or by similar derivatization of other phosphorothioate ODNs developed for the immune activation of rodent or human cells. The in vivo antitumor efficacy of the immunostimulatory H1826 and H2006 ODNs was compared with that of G3139. Bcl-2 suppression achieved by G3139 purportedly sensitizes tumor cells toward cytotoxic agents, and some of the experiments employed combinations of ODN with such drugs as cisplatin or etoposide. H1826, H2006, and G3139 all produced similar, striking, growth inhibitory effects on either H69 SCLC, A2780 ovarian carcinoma, or A549 lung adenocarcinoma human tumor xenografts at doses of 0.3 mg/kg and 1 mg/kg (H1826, H2006) or 12 mg/kg (G3139) per day. In contrast, the H2006-mC (1 mg/kg) or G3139-mC (12 mg/kg) derivatives demonstrated no significant antitumor effects. The combination of G3139 (12 mg/kg) with cisplatin produced some additive antitumor efficacy, which was not seen in combinations of G3139-mC (12 mg/kg) or H1826 (1 mg/kg) with cisplatin. G3139, at a dose of 12 mg/kg, alone induced extensive enlargement of the spleen. Immunostimulation was evaluated in vitro by flow cytometric measurements of the CD80 and CD86 activation markers found on CD19+ murine splenocytes. The CpG-ODN producing strong antitumor effects in vivo also induced these activation markers in vitro, in contrast to the in vivo inactive G3139-mC. Our data indicate a significant contribution of the immunostimulatory properties of CpG-ODN (including G3139) to the antitumor effects observed in nude mouse xenograft models. This is in contrast to previous data presented by other authors indicating that the activity of G3139 in human tumor xenografts was Bcl-2 specific. Furthermore, as nude mice are devoid of T cells, a T cell-mediated immune response apparently is not required for the potent antitumor responses observed here; innate immune responses are sufficient.

Adenocarcinoma↗
PubMed1997

Ex vivo treatment of bone marrow with phosphorothioate oligonucleotide OL(1)p53 for autologous transplantation in acute myelogenous leukemia and myelodysplastic syndrome.

Effective ex vivo purging techniques can decrease the likelihood of infusing bone marrow contaminated with leukemic cells during autologous transplantation. In preliminary studies, OL(1)p53, a 20-mer phosphorothioate oligonucleotide directed against p53 mRNA, decreased the number of acute myelogenous leukemia (AML) cells in vitro, suggesting a possible role for OL(1)p53 in purging bone marrow harvests of leukemia cells. To demonstrate that OL(1)p53 was nontoxic to hematopoietic progenitor cells, normal bone marrow cells were incubated with 10 microM OL(1)p53 for 36 h, and hematopoietic progenitor cell survival was determined by in vitro colony assays. OL(1)p53 had no toxic effect on the growth of either myeloid (CFU-GM) or erythroid (BFU-E) progenitor cells. OL(1)p53 was then used to ex vivo purge bone marrow harvests from nine patients with either AML or myelodysplastic syndrome (MDS). Bone marrow cells were incubated with 10 microM OL(1)p53 for 36 h before transplantation. The median times posttransplantation for the patient to recover an absolute neutrophil count greater than 0.5 x 10(9)/L and a platelet transfusion independence were 30 days and 56 days, respectively. Incubation of bone marrow cells with OL(1)p53 had no detrimental effect on the growth of hematopoietic progenitor cells, and transplantation of autologous bone marrow cells treated with the phosphorothioate oligonucleotide, OL(1)p53, resulted in successful recovery of circulating neutrophils following high-dose therapy in patients with AML or MDS. The data show that OL(1)p53 can be used safely to purge autologous bone marrow harvests from patients with leukemia.

Adult↗
PubMed1985

The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA.

M13 RF IV DNA may be prepared in vitro to contain phosphorothioate-modified internucleotidic linkages in the (-)strand only. Certain restriction enzymes react with this modified DNA to hydrolyze the (+)strand exclusively when a phosphorothioate linkage occurs at the normal cleavage point in the (-)strand. The reaction of Pvu I with M13mp2 RF IV DNA containing dCMPS residues in the (-)strand is of this type, and is exploited to allow subsequent digestion with exonuclease III of a portion of the (+)strand opposite different mutagenic mismatched oligonucleotide primers. Two methods are described by which this approach has been used to produce mutations in M13mp2 phage DNA with high efficiency as a result of simple and rapid in vitro manipulations. Plaques containing mutant phage in a genetically-pure form are obtained at a frequency of 40-66%, allowing their characterisation directly by sequence analysis without prior screening and plaque purification. The wide applicability of this approach is discussed.

DNA↗
PubMed1986

Inhibition of restriction endonuclease Nci I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis.

M13 RF IV DNA where phosphorothioate groups are incorporated at restriction endonuclease Nci I recognition sites in the (-)strand is efficiently nicked by the action of this enzyme. Incubation of such nicked DNA with exonuclease III produces gapped DNA. The gap can be filled by reaction with deoxynucleoside triphosphates and DNA polymerase I. When this sequence of reactions is performed with DNA containing a mismatch oligonucleotide primer in the (-)-strand mutational frequencies of 70-90% can be obtained upon transformation. The general nature of this methodology has been further shown to be applicable to other restriction enzymes such as Hind II, Pst I and Fsp I. The mutational frequency obtained using these enzymes is between 40-80% mainly because of less efficient nicking and gapping. Studies on inhibition of Nci I cleavage show that in addition to a phosphorothioate group at the position of cleavage an additional group in the 5'-neighbouring position is necessary for complete inhibition.

DNA Polymerase I↗
PubMed1987

Stereospecificity of nucleases towards phosphorothioate-substituted RNA: stereochemistry of transcription by T7 RNA polymerase.

Transcription by T7 RNA polymerase has been studied using a chiral ATP analogue. The Sp diastereoisomer of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) was incorporated into RNA with an apparent KM of approximately 15 microM, similar to that for ATP; the Rp diastereoisomer was neither a substrate nor a competitive inhibitor. The configuration of the phosphodiester link in the RNA produced was analyzed with stereospecific nucleases. The rate of nuclease digestion was compared with the rate of digestion of phosphorothioate-substituted RNA of known stereochemistry synthesized by E. coli RNA polymerase. Surprisingly, the nucleases exhibited reduced discrimination compared with their activity on dinucleotides. The results show that phosphorothioate-substituted RNA transcribed by T7 RNA polymerase has the same configuration as that transcribed by E. coli RNA polymerase, ie. Rp. Thus, the reaction proceeds with inversion of configuration at phosphorus.

DNA-Directed RNA Polymerases↗
PubMed1987

Comparative inhibition of chloramphenicol acetyltransferase gene expression by antisense oligonucleotide analogues having alkyl phosphotriester, methylphosphonate and phosphorothioate linkages.

Several classes of oligonucleotide antisense compounds of sequence complementary to the start of the mRNA coding sequence for chloramphenicol acetyl transferase (CAT), including methylphosphonate, alkyltriester, and phosphorothioate analogues of DNA, have been compared to "normal" phosphodiester oligonucleotides for their ability to inhibit expression of plasmid-directed CAT gene activity in CV-1 cells. CAT gene expression was inhibited when transfection with plasmid DNA containing the gene for CAT coupled to simian virus 40 regulatory sequences (pSV2CAT) or the human immunodeficiency virus enhancer (pHIVCAT) was carried out in the presence of 30 microM concentrations of analogue. For the oligo-methylphosphonate analogue, inhibition was dependent on both oligomer concentration and chain length. Analogues with phosphodiester linkages that alternated with either methylphosphonate, ethyl phosphotriester, or isopropyl phosphotriester linkages were less effective inhibitors, in that order. The phosphorothioate analogue was about two-times more potent than the oligo-methylphosphonate, which was in turn approximately twice as potent as the normal oligonucleotide.

Acetyltransferases↗
PubMed1988

An intermediate in the phage lambda site-specific recombination reaction is revealed by phosphorothioate substitution in DNA.

It has been proposed that phage lambda site-specific recombination proceeds via two independent strand exchanges: the first exchange forming a Holliday-structure which is then converted into complete recombinant products by the second strand exchange. If this hypothesis is correct, one should be able to trap the putative Holliday intermediate by preventing the second strand exchange. In this paper, we show that substitution of phosphorothioate for phosphate in one strand of a recombination site is an effective way to block recombination while permitting the accumulation of a novel structure. This effect is seen only when phosphorothioate is positioned at a point of potential cleavage by Int recombinase, demonstrating that the inhibition of strand exchange is highly specific. Analysis of the novel structure that accumulates in these reactions proves that it contains a Holliday joint. Holliday-structures can also be detected in unblocked recombinations but are present at very low levels. The characteristics of Holliday-structure formation that we describe substantiate the proposed recombination pathway.

Bacteriophage lambda↗
PubMed1988

5'-3' exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis.

The application of T7 and lambda exonuclease to phosphorothioate-based oligonucleotide-directed mutagenesis was investigated. Oligonucleotide primers designed to introduce single or double base mismatches, an insertion or a deletion (each of 16 bases) were annealed to M13 phage derivatives. Double stranded closed circular DNA (RF IV) containing phosphorothioate internucleotidic linkages in the (-)strand was prepared enzymatically from these templates. A nick was introduced into the (+)strand of the hetroduplex DNA. This nicked DNA (RF II) was subjected to treatment with T7 or lambda exonuclease. Both of these enzymes were able to degrade almost all of the viral (+)strand when presented with DNA containing one or two base mismatches. Repolymerisation of the DNA after the gapping reaction, followed by transfection into E. coli cells gave mutational efficiencies of up to 95%. In the case of RF II DNA prepared with insertion or deletion primers these exonucleases could only partially degrade the viral (+)strand but were nevertheless highly efficient in such mutagenesis experiments.

Base Composition↗
PubMed1988

Physicochemical properties of phosphorothioate oligodeoxynucleotides.

We have recently shown that phosphorothioate (PS) oligodeoxynucleotide (ODN) analogs, unlike their normal congeners, exhibit significant anti-HIV activity (Matsukura et al., (1987) Proc. Natl. Acad. Sci. USA 84, 7706-7710). We now report the syntheses, melting temperatures (Tm), and nuclease susceptibilities of a series of phosphorothioate ODN analogs. These include all-PS duplexes, duplexes with one normal chain and the other chain either all-PS, or end-capped with several PS groups at both 3' and 5' ends. The DNase susceptibilities of the S-ODNs are much less than the normal phosphodiesters, but by contrast duplexes of poly-rA with S-dT40 are much more susceptible to RNase H digestion. The Tm's for AT base pairs of S-ODNs are significantly depressed relative to normals, while GC base pairs 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 have significance for the biological properties of these analogs as anti-message inhibitors of gene expression, and provide a rational basis for the S-dC/G sequences as potential effective anti-AIDS agents.

Chemical Phenomena↗
PubMed1989

RNase H cleavage of RNA hybridized to oligonucleotides containing methylphosphonate, phosphorothioate and phosphodiester bonds.

Three types of 14-mer oligonucleotides were hybridized to human beta-globin pre-mRNA and the resultant duplexes were tested for susceptibility to cleavage by RNase H from E. coli or from HeLa cell nuclear extract. The oligonucleotides contained normal deoxynucleotides, phosphorothioate analogs alternating with normal deoxynucleotides, or one to six methylphosphonate deoxynucleosides. Duplexes formed with deoxyoligonucleotides or phosphorothioate analogs were susceptible to cleavage by RNase H from both sources, whereas a duplex formed with an oligonucleotide containing six methylphosphonate deoxynucleosides alternating with normal deoxynucleotides was resistant. Susceptibility to cleavage by RNase H increased parallel to a reduction in the number of methylphosphonate residues in the oligonucleotide. Stability of the oligonucleotides in the nuclear extract from HeLa cells was also tested. Whereas deoxyoligonucleotides were rapidly degraded, oligonucleotides containing alternating methylphosphonate residues remained unchanged after 70 minutes of incubation. Other oligonucleotides exhibited intermediate stability.

Base Sequence↗
PubMed1991

Configurationally defined phosphorothioate-containing oligoribonucleotides in the study of the mechanism of cleavage of hammerhead ribozymes.

The chemical synthesis is described of oligoribonucleotides containing a single phosphorothioate linkage of defined Rp and Sp configuration. The oligoribonucleotides were used as substrates in the study of the mechanism of cleavage of an RNA hammerhead domain having the phosphorothioate group at the cleavage site. Whereas the Rp isomer was cleaved only very slowly in the presence of magnesium ion, the rate of cleavage of the Sp isomer was only slightly reduced from that of the unmodified phosphodiester. This finding gives further evidence for the hypothesis that the magnesium ion is bound to the pro-R oxygen in the transition state of the hammerhead cleavage reaction. Also, inversion of configuration at phosphorus is confirmed for a two-stranded hammerhead.

Base Sequence↗
PubMed1991

Synthesis and selective cleavage of an oligodeoxynucleotide containing a bridged internucleotide 5'-phosphorothioate linkage.

A self complementary oligodeoxynucleotide dodecamer containing an achiral bridged 5'-phosphorothioate linkage 3'-O-P-S-5' has been prepared using the solid phase phosphoramidite procedure. For the incorporation of the phosphorothioate link we have used a 5'-(S-trityl)-mercapto-5'-deoxythymidine-3'-phosphoramidite. After coupling this building block the S-trityl group was removed by silver ions. The free thiol moiety was then coupled with a standard phosphoramidite in the presence of tetrazole. After oxidation of the 5'-phosphorothioite with iodine/water the synthesis was continued with standard building blocks up to the desired dodecamer. This backbone modified dodecamer can be cleaved selectively and quantitatively at the P-S bond by silver or mercuric ions under very mild conditions.

Base Sequence↗
PubMed1975

The use of s-2-cyanoethyl phosphorothioate in the preparation of oligo 5'-deoxy-5'-thiothymidylates.

An improvement of our strategy for the stepwise synthesis of oligo 5'-deoxy-5'-thiodeoxyribonucleotides [Chladek and Nagyvary (1972) J. Amer. Chem. Soc. 94, 2079] involves the use of 5'-O-tosylthymidine 3'-S-2-cyanoethyl phosphorothioate. The displacement of the tosylate by thymidine 3'-phosphorothioate and subsequent alkaline deblocking afforded the dinucleotide (Tps)2. The process of displacement and deblocking was repeated three more times at an average yield of 30 percent per step. The corresponding bifunctional derivative of deoxyadenosine was found much less reactive and practically unsuitable for repeated chain elongation. The ORD and CD spectra of the analogs are similar to those of the natural oligonucleotides.

Chromatography, DEAE-Cellulose↗