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PubMed1986

Similarity of isozyme pattern of serum lactate dehydrogenase following treatments with O,O,S-trimethyl phosphorothioate and paraquat.

Oral administration of O,O,S-trimethyl phosphorothioate (OOS-TMP) causes delayed mortality and lung injury in rats. The present study was carried out to determine whether serum lactate dehydrogenase (LDH) activity and its isozyme pattern were capable of detecting lung injury and possible other organ damages caused by OOS-TMP. Carbon tetrachloride (CCl4, 1 ml/kg, i.p., 24 h) and paraquat (25 mg/kg, i.p., 48 h) were selected as standard chemicals for liver injury, and lung and kidney injuries, respectively. The activity of serum LDH increased to 4 times above control following treatment of rats with OOS-TMP (20 mg/kg, p.o., 72 h). Treatment with CCl4 increased the activity to 8 times while paraquat only slightly increased the activity. Isoelectrophoresis of serum samples showed that OOS-TMP increased the activities of isozymes LDH 1 and LDH 2. This isozyme pattern was similar to the pattern of paraquat but quite different from the pattern in animals treated with CCl4, where LDH 5 was most predominant, or in control animals. Thus, the isozyme pattern strongly suggests that OOS-TMP causes the similar organ damage evoked by paraquat, lung and kidney.

Administration, Oral↗
PubMed1987

Dysproteinuria induced in rats by O,O,S-trimethyl phosphorothioate.

Administration of a single oral dose of 60 mg/kg O,O,S-trimethyl phosphorothioate (OOS-Me), a malathion impurity, resulted in a substantial increase in the amounts of amino acids along with a change in the nature of proteins excreted in the urine of treated rats. In contrast to control rats, a small increase in albumin and a small decrease in alpha 1-globulin were observed. However, alpha2-, beta- and gamma 1-globulin, which were not detected in the urine of control rats, were found in substantial amounts in the urine of OOS-Me-treated rats. These findings, coupled with observed increases in urinary glucose levels and consistent specific gravity readings of 1.01 even though treated rats were experiencing oliguria, provide evidence for OOS-Me-induced kidney tubule damage.

Albuminuria↗
PubMed1995

Antisense phosphorothioate oligodeoxynucleotides: introductory concepts and possible molecular mechanisms of toxicity.

Over the past 5 years or so, much attention has been given to the possible use of synthetic antisense oligonucleotide analogs as a new class of therapeutic agents that function by sequence-specific inhibition of genetic expression. The basic design concepts which underline this novel approach to drug discovery are briefly described herein, together with some of the chemical, biochemical, and pharmacological aspects of phosphorothioate oligodeoxynucleotides that are first-generation antisense compounds now under clinical investigation. Possible molecular mechanisms of toxicity for this class, and other structural types of antisense compounds are discussed with the hope of stimulating interest in future toxicological studies in this emerging area of drug development.

Gene Expression↗
PubMed1995

Pharmacology and toxicology of phosphorothioate oligonucleotides in the mouse, rat, monkey and man.

Phosphorothioate oligonucleotides (PS-ODN) designed to temporarily modulate selected gene expression have made the journey from bench top to beside in a remarkably short period of time. A PS-ODN with sequence complementary to the p53 mRNA was administered to mice (4 mg/kg subcutaneously), rats (3-300 mg/kg intravenously), monkeys (intravenous infusions for up to 15 days) and humans (up to 0.25 mg/kg/h intravenous infusions for 10 days). These studies demonstrate the PS-ODN provides feasible pharmacokinetic parameters and minimal toxicity.

Animals↗
PubMed1993

Quantitative analysis of phosphorothioate oligonucleotides in biological fluids using fast anion-exchange chromatography.

Phosphorothioate oligonucleotides are potentially useful as anti-viral drugs. Classical DNA extraction methods are not as effective on short single-stranded DNA as with longer double-stranced chains. The classical method of phenol-chloroform extraction followed by ethanol precipitation is difficult to quantify, thus monitoring of the pharmacological disposition of these compounds is subject to error. A method has been devised and validated for extraction and analysis of modified oligonucleotides from biological fluids such as urine and serum based on protein kinase digestion and phenol-chloroform extraction. Due to the high native ultraviolet absorbance of the oligomers, detection limits in the low ppb range were obtained without derivatization.

Chromatography, Ion Exchange↗
PubMed2006

Antisense oligonucleotides: efficient synthesis of 2'-O-methoxyethyl phosphorothioate oligonucleotides using 4,5-dicyanoimidazole. Are these oligonucleotides comparable to those synthesized using 1H-tetrazole as coupling activator?

Multiple 2'-O-methoxyethyl modified phosphorothioate oligonucleotides of 18-20-mer in length were synthesized at various scales using 4,5-dicyanoimidazole (DCI) as coupling activator. Extensive synthetic, analytical (using ion-pair LC-MS), and in vivo pharmacological, toxicological studies showed that oligonucleotides made with DCI and 1H-tetrazole are chemically and biologically equivalent. This extensive study will help the oligonucleotide therapeutic industry to move from using a potentially explosive activator (1H-tetrazole) to a safe activator (DCI).

Animals↗
PubMed2004

Biodistribution of phosphodiester and phosphorothioate siRNA.

Short interfering RNAs (siRNAs) are valuable tools for analyzing protein function in mammalian cell culture. This success has led to high expectations for in vivo and therapeutic applications. However, the pharmacokinetic properties of siRNA are not known. Here we report the biodistribution of a phosphodiester (PO) siRNA duplex and examine the effect of phosphorothioate (PS) linkages. Our findings indicate that biodistribution of siRNA is similar to that for single-stranded antisense oligonucleotides and offer insights for use of siRNA in vivo.

Animals↗
PubMed2005

Characterization of high molecular weight impurities in synthetic phosphorothioate oligonucleotides.

Phosphorothioate oligonucleotides manufactured by standard phosphoramidite techniques using 2'-deoxyadenosine- or 2'-O-(2-methoxyethyl)-5-methylcytosine-loaded solid supports contain branched impurities consisting of two chains linked through the exocyclic amino group of the 3'-terminal nucleoside of one chain and the 3'-terminal hydroxyl group of another via a P(O)SH group. These impurities are not produced when a universal, non-nucleoside derivatized support is used.

5-Methylcytosine↗
PubMed2005

Intraperitoneal administration of phosphorothioate antisense oligodeoxynucleotide against splicing enhancer sequence induced exon skipping in dystrophin mRNA expressed in mdx skeletal muscle.

Antisense oligodeoxynucleotide against the splicing enhancer sequence (SES) in exon 19 of the dystrophin gene have been shown to induce exon 19 skipping and promote the expression of internally deleted dystrophin by correcting the translational reading frame in the cultured Duchenne muscular dystrophy (DMD) myocytes with the deletion of exon 20. Transfection of the antisense oligodeoxynucleotide, therefore, has been proposed as a promising means for therapeutic modification of dystrophin mRNA of DMD, a fatal disorder caused by defects in the dystrophin gene. A systemic delivery method targeting the large number of diseased muscles remains to be established for clinical application of antisense oligodeoxynucleotide. In this study, we investigated capability of oligodeoxynucleotide transfer into the skeletal muscles of mdx mouse, a mouse model of DMD. Thirty-one mer phosphorothioate oligodeoxynucleotide complementary to the SES of dystrophin exon 19 was intraperitoneally administered to mdx mice without any carrier. Histochemical study disclosed that fluorescence-labeled oligodeoxynucleotide appeared in the nuclei of femoral skeletal muscle cell at the second day after injection of 20 mg/kg BW oligodeoxynucleotide, and still visible at 14th day. Reverse transcription (RT)-PCR analysis of dystrophin transcript in these cells disclosed that a proportion of it showed skipping of exon 19 from second to seventh day after injection. These results showed that the intraperitoneally administered oligodeoxynucleotide could be transfected to nucleus of mdx skeletal muscle without any carrier and was able to induce exon skipping in vivo.

Animals↗
PubMed2005

Biodistribution of 68Ga-labelled phosphodiester, phosphorothioate, and 2'-O-methyl phosphodiester oligonucleotides in normal rats.

Antisense oligonucleotides may hybridise with high selectivity to mRNA sequences allowing monitoring of gene expression or inhibition of the manifestation of altered genes inducing diseases. As part of the development of positron emission tomography methods, 17-mer antisense phosphodiester (PO), phosphorothioate (PS) and 2'-O-methyl phosphodiester (OMe) oligonucleotides specific for point mutationally activated human K-ras oncogene were labelled with 68Ga radionuclide via a chelator coupled to the probe. Hybridisation in solution and non-denaturing polyacrylamide gel electrophoresis (PAGE) with a subsequent exposure of the gels was performed to verify the hybridisation ability after labelling. The biodistribution was studied in male Sprague-Dawley rats by injecting 2MBq of 68Ga-oligonucleotides via the tail vein and measuring the organ radioactivity concentration after 20, 60 and 120 min or using whole-body autoradiography with 10 MBq 68Ga-oligonucleotide and 20 min incubation time. Control experiments were performed with 68GaCl3 and 68Ga-chelator complex. The results revealed that 68Ga-labelling did not change the hybridisation abilities of the oligonucleotides. The biodistribution pattern depended on the nature of the oligonucleotide backbone. Bone marrow, kidney, liver, spleen and urinary bladder were the five organs of highest uptake with each oligonucleotide. The PO accumulated highly in the liver, whereas high kidney uptake dominated the PS and OMe patterns. Intact PS and OMe were detected in plasma samples taken 20 and 60 min after injection. This study supplies a base for the further development of 68Ga-labelled oligonucleotides as pharmacokinetic tools and a potential future use for in vivo imaging of gene expression.

Animals↗
PubMed2006

Single-based resolution for oligodeoxynucleotides and their phosphorothioate modifications by replaceable capillary gel electrophoresis.

A replaceable capillary gel electrophoretic (replaceable CGE) method was developed for the separation of two sets of model compounds of single-stranded oligodeoxynucleotide mixtures (18-20 mers), phosphodiester oligodeoxynucleotides (PO-ODNs) and their phosphorothioate modifications (PS-ODNs), with equal sequences differing in a single base. Polyethylene glycol (PEG) 35000 was chosen as the sieving matrix. It was confirmed that PEG polymer solution less influenced resolutions of the PS-ODNs compared with those of the PO-ODNs, while acetonitrile used as an additive in the system improved the separation significantly. It was also noticed that the effect of temperature on separation was much larger than that of denaturant urea.

Acetonitriles↗
PubMed2005

Effects of methyl parathion (o,o-dimethyl o-4-nitrophenyl phosphorothioate) on rat sperm morphology and sperm count, but not fertility, are associated with decreased ascorbic acid level in the testis.

Methyl parathion (MP; o,o-dimethyl o-4-nitrophenyl phosphorothioate) is an organophosphorous pesticide used world wide to spray agricultural crops. The present study was aimed to investigate the genotoxic and cytotoxic effects on male germ cells and their possible relation with testicular ascorbic acid levels. Adult male Wistar rats (n=5/group) received MP at 0, 0.5, or 1 mg/kg (experiments 1 and 2) for 12 days and 0, 0.75 or 1.5 mg/kg (experiment 3) for 25 days (i.p.) everyday at intervals of 24 h. The epididymal sperm count, sperm abnormalities and testicular ascorbic acid levels (by 2,4-dinitrophenyl hydrazine method) were estimated on days 130, 77 and 17 following the last exposure in experiments 1, 2, and 3, respectively. Virgin untreated female rats were mated with treated males from experiments 2 and 3 for a week effective from day 35 to 41 after the first treatment, and fertility indices were measured after the birth of pups. Sperm count was decreased in experiments 2 and 3 (P<0.01), and in all three experiments, the abnormal sperms increased (P<0.001). Concomitantly, the ascorbic acid levels decreased in the testis (P<0.05-0.001; one-way ANOVA and Bonferroni's post hoc test). The body weights of offspring of treated males did not show significant changes from those of the controls, although there were some decreases observed. MP reduced the lactation index in experiment 2 (P<0.001; Chi-square test). The number of pups/parent along with fertility indices showed some numerical decrease but without any statistical significance. The present findings suggest that MP is a weak genotoxic and cytotoxic agent in the rat exposed to human exposure dose-levels, and that these effects, except the fertility are well correlated with decreased ascorbic acid level in the testis. Furthermore, MP-induced changes in the germ cells do not have any significant effects on F1 generation.

Animals↗
PubMed2001

Increased efficacy of antileishmanial antisense phosphorothioate oligonucleotides in Leishmania amazonensis overexpressing ribonuclease H.

Ribonuclease H (RNase H), an enzyme that cleaves an RNA sequence base-paired with a complementary DNA sequence, is proposed to be the mediator of antisense phosphorothioate oligonucleotide (S-oligo) lethality in a cell. To understand the role of RNase H in the killing of the parasitic protozoan Leishmania by antisense S-oligos, we expressed an episomal copy of the Trypanosoma brucei RNase H1 gene inside L. amazonensis promastigotes and amastigotes that constitutively express firefly luciferase. Our hypothesis was that S-oligo-directed degradation of target mRNA is facilitated in a cell that has higher RNase H activity. Increased inhibition of luciferase mRNA expression by anti-luciferase S-oligo and by anti-miniexon S-oligo in these stably transfected promastigotes overexpressing RNase H1 was correlated to the higher activity of RNase H in these cells. The efficiency of killing of the RNase H overexpressing amastigotes inside L. amazonensis-infected macrophages by anti-miniexon S-oligo was higher than in the control cells. Thus, RNase H appears to play an important role in the antisense S-oligo-mediated killing of Leishmania. Chemical modification of S-oligos that stimulate RNase H and/or co-treatment of cells with an activator of RNase H may be useful for developing an antisense approach against leishmaniasis. The transgenic Leishmania cells overexpressing RNase H should be a good model system for the antisense-mediated gene expression ablation studies in these parasites.

Animals↗
PubMed2001

Hepatic distribution of a phosphorothioate oligodeoxynucleotide within rodents following intravenous administration.

The pharmacokinetics of ISIS 1082, a 21-base heterosequence phosphorothioate oligodeoxynucleotide, were characterized within rodent whole liver, and cellular and subcellular compartments. Cross-species comparisons were performed using Sprague-Dawley rat and CD-1 mouse strains. Although whole liver oligonucleotide deposition and the proportion of drug found within parenchymal and nonparenchymal cells were similar between the two rodent species as a function of both time and dose, dramatic differences in subcellular pharmacokinetics were observed. Specifically, within murine hepatocyte nuclei, drug was observed at the 10 mg/kg dose, whereas in the rat nuclear-associated levels required the administration of 25 mg/kg. Under all experimental regimens, murine hepatic nuclear-associated drug concentrations were at least 2-fold higher than those found in rat liver cells. More detailed metabolic analysis was also performed using high performance liquid chromatography/electrospray-mass spectrometry (HPLC/ES-MS) and demonstrated that although the extent of metabolism was similar for rat and mouse, the pattern of n-1 metabolites varied as a function of both species and cell type. While rat and mouse hepatocytes and rat nonparenchymal cellular metabolites were predominantly products of 3'-exonuclease degradation, mouse nonparenchymal cells contained a majority of n-1 metabolites produced by 5'-exonucleolytic activity. Based upon these data, it would appear that subcellular oligonucleotide disposition and metabolism among rodent species are more divergent than whole organ pharmacokinetics might predict.

Animals↗
PubMed2001

Enhanced activity of antisense phosphorothioate oligos against leishmania amastigotes: augmented uptake of oligo, ribonuclease H activation, and efficient target intervention under altered growth conditions.

Leishmania, a parasitic protozoan, infects human macrophages, often causing severe morbidity and mortality. The pathogenic form of this parasite, the amastigote, lives inside the acidic phagolysosomes of infected macrophages. In our attempt to develop anti-miniexon phosphorothioate oligodeoxyribonucleotides (S-oligos) as an alternative chemotherapy against Leishmania, we found that intracellular as well as 'axenic' amastigotes were more susceptible to these S-oligos than were the cultured promastigotes. Lower pH (4.5) and elevated temperature (35 degrees) of the medium were among the direct enhancing factors for killing. Addition of the cationic polypeptide poly-l-lysine (PLL) to the growth medium further enhanced the killing effect of the S-oligo at pH 4.5. The enhancement of specific ablation of mRNA expression was directly correlated to the increased leishmanicidal activity of the S-oligo. This was shown by the increased inhibition of luciferase activity expressed in transgenic Leishmania amazonensis promastigotes by anti-miniexon S-oligo or anti-luciferase S-oligo at acidic pHs and in the presence of PLL. The leishmanicidal effects of S-oligos at acidic pH and in the presence of PLL were related to increased uptake of the S-oligos under these conditions. The rate of S-oligo uptake was enhanced up to 15-fold at pH 4.5. The addition of PLL to the assay medium at acidic pH further enhanced the uptake of S-oligo up to 80-fold. RNase H is known to accentuate the antisense action of S-oligos. We found that at an elevated temperature RNase H activity in Leishmania cell extracts increased about 5-fold. Thus, enhanced uptake of S-oligos at the acidic pH of macrophage phagolysosomes and activation of RNase H may explain the efficient killing of the parasite in macrophages, both in tissue culture and in the animal model, by antisense miniexon oligonucleotide/PLL, when targeted directly to the parasite-containing phagolysosomes.

Animals↗
PubMed1997

Pharmacokinetics, metabolism, and elimination of a 20-mer phosphorothioate oligodeoxynucleotide (CGP 69846A) after intravenous and subcutaneous administration.

The pharmacokinetics, tissue distribution and metabolism of CGP 69846A, a 20-mer phosphorothioate oligodeoxynucleotide targeted against the 3'-untranslated region of human c-raf-1 kinase mRNA, were investigated in vivo in rats after intravenous and subcutaneous administration. Intravenous disposition studies with [3H]CGP 69846A were supported with analysis by capillary gel electrophoresis and electrospray mass spectrometry. In combination, these techniques provide a detailed account of the pharmacokinetic and metabolic profile for this compound. The elimination of CGP 69846A after a single intravenous dose was studied over extended periods in mice using whole-body autoradiography and capillary gel electrophoresis. Subcutaneous administration to rats resulted in a significant bioavailability with peak plasma levels 4.5-fold lower than after intravenous dosing. This dose route resulted in low interanimal variability and only slightly greater metabolism of the oligonucleotide compared to the intravenous administration.

Animals↗
PubMed1998

Sequence dependency of the internalization and distribution of phosphorothioate oligonucleotides in vascular smooth muscle cells.

Antisense studies imply the utilization of oligonucleotides (ODN) for sequence-specific down-regulation of genes. This usually consists in assessing antisense sequences versus control sequences (mismatched, inverted, scrambled, randomized or any sequence unrelated to the relevant target). Even though the investigated biological effect (knockdown of an unwanted protein) is observed only with the antisense sequence and weakly, if at all, with any of the control sequences, this is a necessary but not a sufficient condition to demonstrate an antisense effect. Indeed, biochemical parameters such as stability, uptake and subcellular compartmentalization of ODN in a given cellular system are most often sequence-dependent processes. In this work, a series of phosphorothioate ODN of different lengths and sequences were evaluated as to their binding, internalization and subcellular distribution properties in vascular smooth muscle cells. In addition to membrane binding and nuclear accumulation, the partition of ODN in the cytosol of cells was measured by a method based upon controlled permeabilization of the plasma membrane, permitting the recovery of the cytosolic content with minimal damage to the membranes of the endocytic vesicles and lysosomes. We found that the tested ODN showed striking differences in their uptake and distribution in smooth muscle cells. Our results gave rise to the problem of validating the observed biological effects when different sequences of ODN were compared. Cellular studies such as the one presented in this work could help in choosing the proper control sequences among ODN exhibiting similar cell interactions as compared to the antisense sequences. Moreover, this method could be useful for the selection of antisense sequences that can be efficiently internalized and preferentially distributed in the appropriate compartments in cells for in vitro antisense studies.

Animals↗