Mutagenesis, Vol. 17, No. 3, 201-209,
May 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
Assessment of the genotoxic potential of ISIS 2302: a phosphorothioate oligodeoxynucleotide
ISIS Pharmaceuticals, Carlsbad Research Center, 2292 Faraday Avenue, Carlsbad, CA 92008, USA
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Abstract |
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ISIS 2302, a phosphorothioate oligodeoxynucleotide with antisense activity against human ICAM-1 mRNA, was evaluated in a battery of tests to assess genotoxic potential. There was no evidence of genotoxicity in three in vitro studies performed: (i) a bacterial reverse mutation test; (ii) a chromosomal aberration test in Chinese hamster ovary cells; (iii) a mammalian cell gene mutation assay in L5187Y cells. Additionally, there was no in vivo evidence of genetic toxicity in a bone marrow micronucleus study in male and female mice. For all tests, top concentrations or doses assessed met harmonized regulatory guidelines. The cellular uptake of ISIS 2302 into target cells was confirmed using capillary gel electrophoresis and immunohistochemistry. Intracellular uptake into CHO cells, L5187Y cells, Salmonella typhimurium TA98 and bone marrow was concentration- and time-dependent. Consistent with what is known about the physical and chemical properties of phosphorothioate oligodeoxynucleotides, there was no evidence of genotoxicity in any of the assessed end-points. Furthermore, the absence of genotoxicity could not be ascribed to test system insensitivity or to an absence of exposure of the test system to ISIS 2302.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Phosphorothioate oligodeoxynucleotides designed to hybridize to the mRNAs of specific target genes can inhibit expression of that gene through an antisense mechanism of action (Crooke, 1992
). Binding of the oligodeoxynucleotide to its target mRNA to form a duplex results in the activation of RNase H and degradation of the target mRNA. This activity is being utilized to produce therapeutically active oligodeoxynucleotides that reduce the expression of genes that are inappropriately expressed in disease states (Wagner, 1994; Crooke, 1995). The compound employed in the present studies, ISIS 2302, is a 20 base phosphorothioate oligodeoxynucleotide complementary to the 5'-untranslated region of the human ICAM-1 mRNA that has been shown to reduce mRNA expression in human cell lines (Bennett, 1994). Inhibition of ICAM-1 expression may help modulate a number of inflammatory diseases because of its role in the margination and trafficking of leukocytes at sites of inflammation, as well as its role in T cell activation.
ISIS 2302 is currently being developed as a therapeutic agent for Crohn's disease (Glover et al., 1997; Yacyshyn et al., 1998) and has shown activity in disease models associated with chronic inflammation (Bennett, 1993, 1994; Stepkowski et al., 1994). As part of the characterization of the toxicity of this compound we have performed several in vitro and in vivo genotoxicity tests. ISIS 2302 (molecular weight 
7 kDa) differs from endogenous DNA by virtue of substitution of sulfur for one of non-bridging oxygens in each of the phosphate linkages (Figure 1). This chemical modification increases the nuclease resistance of the synthetic oligodeoxynucleotide compared with endogenous DNA and increases the affinity of the oligodeoxynucleotide for proteins (Hoke et al., 1991). Oligodeoxynucleotides of this class are highly charged and hydrophilic, suggesting that cellular uptake by simple diffusion across cell membranes could be limited. Studies on the uptake of oligodeoxynucleotides by cells in culture have demonstrated that uptake is both time- and temperature-dependent (Takakura et al., 1996, 1998; Wingens et al., 1998). In vivo oligodeoxynucleotides appear to readily enter a number of different cell types, but the mechanism of uptake is not fully understood (Butler et al., 1997a; Graham et al., 1998).
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In addition to evaluating the genotoxic potential of ISIS 2302, we were interested in determining if the oligodeoxynucleotide could be detected in cells under conditions similar to those used in routine genotoxicity testing. By characterizing the presence of oligodeoxynucleotide within target cells, the question of the validity of performing cell-based genotoxicity assays with molecules of this class can be established. The results clearly demonstrate that ISIS 2302 is internalized and metabolized by the test cell and tissue types, but that genotoxicity was not observed.
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Materials and methods |
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Bacterial reverse mutation test
The bacterial reverse mutation test uses amino acid-requiring strains of Salmonella typhimurium and Escherichia coli to detect point mutations that involve substitution, addition or deletion of one or a few DNA base pairs. The bacterial reverse mutation test is widely used and many of the test strains have features that make them more sensitive for the detection of mutations. ISIS 2302 was evaluated in a bacterial reverse mutation test using S.typhimurium strains TA98, TA100, TA1535 and TA1537 and in E.coli strain WP2 uvrA using standard plate incorporation methods (Ames et al., 1975
; Maron and Ames, 1983; Gatehouse et al., 1994). In comparison with concurrent controls, a range of concentrations up to 5000 µg/plate ISIS 2302 were assessed in the presence and absence of an exogenous metabolic activation system. The metabolic activation system used was the cofactor-supplemented post-mitrochondrial fraction (S9) prepared from the livers of adult male Sprague–Dawley rats treated with Aroclor 1254. Appropriate positive controls were used for each tester strain and metabolic activation condition. A positive response would require a significant and dose-dependent increase in the number of cultures that reverted to amino acid independence.
Exposure assessment
For the analysis of oligodeoxynucleotide uptake in bacteria, S.typhimurium strain TA98 was selected as being representative of the other tester strains. An overnight culture of TA98 was suspended at 109 cells/ml in a 10% glucose minimal medium with Vogel-Bonner salts, histidine, biotin and sodium phosphate buffer (normal top agar ingredients without agar) and aliquoted into 50 ml conical tubes. ISIS 2302 in phosphate-buffered saline (PBS) was added to 0.5 ml of S9 mix or buffer to yield concentrations of 500 or 5000 µg/ml. Control incubations received equal volumes of the PBS vehicle. Solutions were mixed and incubated at 37°C for 24 or 48 h. Three cultures per dose level per time point in the presence and absence of the exogenous metabolic activation system were used. Following exposure, cells were harvested by centrifugation (3000 g for 10 min) and a 1 ml aliquot of medium was removed and frozen for oligodeoxynucleotide analysis. The remaining medium was removed and the cell suspension washed twice with ice-cold medium. After the second wash, the pelleted cells were resuspended in 3 ml of proteinase K buffer. Cell suspensions were transferred to microcentrifuge tubes, frozen immediately on dry ice and stored at –80°C until analysis by capillary gel electrophoresis (CGE).
Mammalian chromosomal aberration assay in Chinese hamster ovary cells
The in vitro chromosome aberration test employs cultures of established cell lines, strains or primary cell cultures to assess the potential of agents to induce structural chromosomal aberrations (Evans, 1976; Galloway et al., 1994). ISIS 2302 was evaluated in an in vitro chromosome aberration test at concentrations up to 5000 µg/ml both with (activated assay) and without (non-activated assay) an exogenous S9 metabolic activation system. Chinese hamster ovary cells (CHO) were obtained from Dr Sheila Galloway (Merck Institute for Therapeutic Research, West Point, PA). The cell line had a modal chromosome number of 21 and doubling time of 13–14 h in the medium and under the conditions used for testing. Prior to use, cell stocks were checked and found to be free of Mycoplasma contamination. All exposures were performed in duplicate flasks. CHO cells were seeded at a density of 7.5x105 cells/25 cm flask and following overnight incubation were continuously exposed to the test compound for 14 or 20 h in the non-activated test system (–S9 mix). In the activated assay, the exposure period was 2 h in serum-free medium, followed by washing and a growth period of 14 or 20 h as described for the non-activated assay. Cell harvesting and slide preparation followed standard cytogenetic procedures involving hypotonic treatment, fixation and staining. A positive response would require a significant and dose-dependent increase in the number of aberrations.
Exposure assessment
To demonstrate cellular uptake of oligodeoxynucleotide in CHO cells, separate experiments were performed under conditions representative of those used for the assessment of genotoxic potential. In triplicate and with appropriate concurrent controls, ISIS 2302 was added at 500 or 5000 µg/ml to 2x106 cells/culture in 5 ml of McCoy's 5A medium. Cells were incubated at 37°C for 20 h as in the non-activated assay (–S9 mix) and 2 h as in the activated assay (+S9 mix). At the end of the exposure, 1 ml aliquots of medium were removed and immediately frozen on dry ice. The remaining medium was removed and the cell monolayer washed twice with ice-cold, serum-free medium. Following the second wash, the cells were removed by trypsinization and then transferred to a centrifuge tube. An aliquot of the cell suspension was removed and the number of viable cells determined by trypan blue exclusion. A 1 ml aliquot of medium was removed and frozen immediately for analysis of oligodeoxynucleotides. Cells were washed in serum-free medium, viable cells enumerated and cell pellets were resuspended in 2 ml of proteinase K buffer at a cell density of 0.5–1.0x106 cells/ml and incubated for 2 h. An aliquot of the cell suspension (106 cells) was frozen as described above prior to analysis.
In vitro mammalian cell gene mutation test in L5178Y cells
The mouse lymphoma cell mutagenesis assay has been widely used to detect and quantitate the induction of mutations in mammalian cells (Clive et al., 1983,1987, 1995). ISIS 2302 was evaluated for mutagenic activity in the presence and absence of an exogenous S9 metabolic activation system. Concentrations of up to 5000 µg/ml were assessed in initial and independent confirmatory experiments. L5178Y mouse lymphoma cells (clone 3.7.2.C) were obtained from Dr D.Clive (Burroughs Wellcome, NC) and cultivated in RMPI 1640 medium supplemented with 0.1% Pluronic F68, 0.2 mg/ml sodium pyruvate (R0P) and 10% heat-inactivated donor horse serum (R10P). In both the initial and confirmatory assays L5178Y cells were incubated at concentrations of 0, 500, 1000, 2000, 4000 and 5000 µg/ml ISIS 2302 for 4 h. The cell culture medium was then changed and the cells were incubated for an additional 2 days without ISIS 2302. The frequency of trifluorothymidine-resistant (TFTr) cells, relative toxicity and average frequency of small and large TFTr colonies in each set of replicate cell cultures were calculated. Upon completion of the first experiment, a complete independent confirmatory experiment in the presence and absence of S9 mix was conducted using the routine techniques as referenced above. A positive response would require a significant and dose-dependent increase in the mutation frequency.
Exposure assessment
In triplicate and in comparison with vehicle controls, ISIS 2302 at 500 and 5000 µg/ml was added to 6x106 L5178Y cells/culture in RPMI 1640 medium and incubated at 37°C for 4 h in the presence and absence of S9 mix. At the end of the exposure period, the cells were separated from the medium by centrifugation (1000 g for 5 min) and 1 ml aliquots of medium were removed and frozen for analysis. The remaining medium was removed and the pellet resuspended in ice-cold medium. Cells were then centrifuged and resuspended again in ice-cold medium. A 1 ml aliquot was removed and added to a vial containing 9 ml of isotonic saline for determination of cell number using a Coulter counter. An aliquot of the suspension containing 5x106 cells was centrifuged, the medium removed and the pellet resuspended in 5.0 ml of proteinase K buffer at a concentration of 1x106 cells/ml. This cell suspension was then transferred to microcentrifuge tubes (1 ml/tube) and frozen as described above prior to analysis by CGE.
Bone marrow mouse micronucleus test
The in vivo micronucleus test is used to identify substances that cause cytogenetic damage to the chromosomes or the mitotic apparatus of cells that results in the formation of micronucleated cells containing lagging chromosome fragments of whole chromosomes (Mavournin et al., 1990). Bone marrow is routinely used, as it is a highly proliferative tissue and the site of production of polychromatic erythrocytes that are easily differentiated from the majority of anucleated peripheral blood cells.
Based on previous experience, Swiss Webster mice were treated in a preliminary range-finding experiment with 600, 800 or 1000 mg/kg ISIS 2302 in PBS, pH 7.4, and observed for clinical signs daily thereafter for 7 days. No lethality occurred at any administered dose, however, dose-related clinical signs were present in both sexes at 800 and 1000 mg/kg. Based on these observations, an experiment was performed with a top dose of 1000 mg/kg. Lower doses of 500 and 250 mg/kg (half and a quarter of the top dose) were included to complete the dose range. In the definitive experiment, 15 mice per sex were treated with a single i.v. injection of 5, 10 or 20 ml/kg body wt of a 50 mg/ml solution of ISIS 2302 that yielded doses of 250, 500 and 1000 mg/kg. Four extra male mice and one extra female mouse were added to the top dose groups of 1000 mg/kg as a precaution for unexpected mortality. A concurrent positive control group of five males was dosed with 25 mg/kg cyclophosphamide and concurrent control groups of 15 male and female mice were administrated saline vehicle at a dosing volume of 20 ml/kg.
Body weight and clinical observations were recorded daily throughout the duration of the study. Four or five mice per sex per dose group were killed at 24, 48 or 72 h post-dosing. Bone marrow was obtained from femurs immediately following scheduled killing. The collected marrow was processed and cells transferred to labeled microscope slides, spread, air dried and fixed in methanol. Slides were coded, stained with acridine orange (Hayashi et al., 1983) and evaluated for the presence of micronuclei by epifluoresence microscopy at a magnification of 630x.
Exposure assessment
Male Swiss Webster mice were administered a single i.v. injection (10 ml/kg body wt) of ISIS 2302, yielding doses of 250 and 1000 mg/kg. At time points of 4, 24, 48 and 72 h, groups of five mice per dose were killed and plasma, bone marrow and sternum collected. A 4 h time point was included as this is near the estimated time of peak tissue concentration (Cmax) for phosphorothioate oligodeoxynucleotides (Cossum et al., 1993; Geary et al., 1997). Blood was collected in tubes containing EDTA and the plasma obtained by subsequent centrifugation was immediately frozen and stored at –70°C until further extraction and analysis. Bone marrow from both femurs was aspirated using syringe needles and fetal bovine serum (FBS) containing 1.7 mg/ml sodium EDTA. Approximately 0.5 ml of FBS was used to collect aspirates of bone marrow into pre-weighed tubes maintained on ice. Following centrifugation, the supernatant was removed and the weight of samples determined. All samples collected were stored at –70°C until extraction and analysis of oligodeoxynucleotides by CGE.
Immunohistochemistry experiments
Sternums collected at necropsy for the mouse micronucleus study were fixed in 10% phosphate-buffered formalin and decalcified overnight using BBC Regularcal Immuno decalcifying agent. Tissue was placed in 70% ethanol until dehydration and paraffin embedding. Paraffin sections were cut and subsequently processed for immunohistochemical staining for oligodeoxynucleotide and evaluation by microscopy as previously described (Butler et al., 1997a,b). Immunohistochemical detection of intracellular oligodeoxynucleotides in collected bone marrow cell aspirates was also conducted as described for in vitro culture systems.
Immunohistochemical detection of intracellular oligodeoxynucleotides in CHO and L5178Y cells was conducted in separate experiments. Cells were treated under conditions similar to those used for the individual genotoxicity tests. Following exposure to vehicle or 500 or 5000 µg/ml ISIS 2302 in the presence or absence of S9 mix, cells were rinsed twice in PBS and fixed in 4% paraformaldehyde for 15 min at room temperature. Cells were then rinsed with PBS and treated with methanol for 10 min. After air drying, cells were rehydrated in PBS and blocked with 5% normal donkey serum (NDS) in PBS for 10 min. Cells were incubated for 1 h with a primary antibody (mouse derived) that is specific for phosphorothioate oligodeoxynucleotides diluted at 1:1000 in 1% NDS and PBS. Following incubation, cells were rinsed with PBS and incubated with a secondary antibody, donkey anti-mouse horseradish peroxidase from Jackson Laboratories (1:100), for 30 min. To visualize peroxidase activity, 3,3'-diaminobenzidine (Sigma, St Louis, MO) was used. Sections were counterstained with hematoxylin and mounted in permanent mounting medium.
Capillary gel electrophoresis analyses
All samples were analyzed by methods similar to those previously published for the analysis of ISIS 2302 in plasma (Leeds et al., 1996). Briefly, a known amount of internal standard (a 27mer polythymidine oligodeoxynucleotide, T27) was added to an aliquot containing a known number of cells (CHO, L5187Y or TA98 cells collected from medium incubations). The cells were then digested with proteinase K at 55°C for 1 h. Following digestion, the aliquot was extracted with phenol/chloroform. The aqueous layer was then further extracted using a two-step solid phase extraction method. The first step involved loading the aliquot onto a strong anion exchange column (Isolute) followed by washing and then eluting the oligodeoxynucleotides with a strong salt buffer solution. The eluted oligodeoxynucleotide solution was loaded onto a reverse phase column (Isolute C18), washed to remove salts and then recovered with 20% acetonitrile in water. The eluate was then evaporated to dryness and reconstituted in water. The reconstituted extracts were then injected electrokinetically onto a Beckman PACE/5000 capillary electrophoresis instrument (CGE) equipped with a gel-filled capillary column and a UV detector set at 260 nm.
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Results |
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Bacterial reverse mutation test
The bacterial mutagenicity of ISIS 2302 was evaluated using standard plate incorporation procedures. Studies were performed in the presence and absence of metabolic activation (rat liver S9) and results were confirmed in independent trials at concentrations up to 5000 µg/plate. No increase from background mutation frequency was observed in any of the tester strains incubated with ISIS 2302 (data not shown). ISIS 2302 was judged negative in initial and confirmatory trials.
To establish that bacteria in these experiments were exposed to the oligodeoxynucleotides, a parallel experiment was performed under nearly identical conditions to those used in the assessment of bacterial mutagenicity. Salmonella typhimurium tester strain TA98 was selected for these experiments. Estimated concentrations of ISIS 2302 in the TA98 strain following incubation for 24 and 48 h with 500 and 5000 µg/ml in the absence of S9 mix ranged from 35 to 238 µM, respectively (Table I). The intracellular concentrations of ISIS 2302 were time- and concentration-dependent. In addition, it was possible to detect the presence of metabolites of ISIS 2302. These metabolites consisted of oligodeoxynucleotide derivatives that were shortened by the cleavage of single nucleotides. A pattern of metabolites where the parent 20mer, the 19mer, 18mer and 17mer are present in progressively decreasing concentrations was observed. This pattern is consistent with metabolic degradation by ubiquitous exonucleases. However, there was no apparent increase in the extent of metabolism by the S9 fraction
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Mammalian chromosomal aberration assay in Chinese hamster ovary cells
ISIS 2302 was evaluated for the ability to induce chromosomal aberrations using Chinese hamster ovary cells in vitro. In the non-activated assay using continuous treatment for 14 h, there was a significant dose-related reduction in the mitotic index to 11% of the concurrent control (Table II
). A similar dose-related depression in mitotic index was observed with continuous treatment in the 20 h assay, but the overall cell number and confluence of the cells in culture were unaffected. In the activated assays there was no evidence of a concentration-related reduction in mitotic index. Time- and concentration-dependent uptake of ISIS 2302 was demonstrated in CHO cells in both the activated and non-activated trials (Table II). There was a slight increase in the number of cells with aberrations in the 14 h activated and non-activated assays that was not present when cells were observed in the 20 h assays. However, these were within the range of control values (0–5% chromosomal aberrations) for the performing laboratory (Biodevelopment Laboratories Inc., Cambridge, MA) and were not statistically significant (Margolin et al., 1986) in comparison with vehicle controls. In the non-activated assay after 20 h incubation it was not possible to analyze the response at 5000 µg/ml because the numbers of mitoses were greatly diminished (<0.3%). Again, ISIS 2302 treatment was not grossly cytotoxic at 5000 µg/ml, as there was no notable decrease in the number or confluence of cells.
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In the 14 and 20 h activated assays no toxicity was noted and mitotic indices were similar to vehicle control values. ISIS 2302 did not cause a significant increase in the percentage of cells with aberrations when compared with controls in either the 14 or 20 h assay at any of the concentrations evaluated. The positive controls, mitomycin C in the non-activated assays and cyclophosphamide in the activated assays, all induced significant chromosomal damage, demonstrating the efficacy of the test system to detect clastogens.
Incubation of CHO cells for 20 h resulted in significant intracellular uptake of ISIS 2302 that was concentration and time dependent (Table I
). Both full-length oligodeoxynucleotide and significant concentrations of metabolites were present in cell extracts. The metabolites were shortened versions of ISIS 2302, with the 19mer being the most abundant and the 17mer least abundant (data not shown). Of the total oligodeoxynucleotide measured in cell extracts, 38 and 42% were in the form of metabolites at the low and high concentrations, respectively. Oligodeoxynucleotide was also detected in CHO cells exposed to ISIS 2302 for 4 h, however, concentrations and the extent of metabolism were less than that seen in the 20 h incubation. There was no apparent effect of S9 mix on metabolism of ISIS 2302. A monoclonal antibody directed against phosphorothioate oligodeoxynucleotides was used to demonstrate cellular internalization. Positive staining for oligodeoxynucleotide in cells exposed to ISIS 2302 was observed (Figure 2).
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In vitro mammalian cell gene mutation test in L5178Y cells
ISIS 2302 was assessed for cytotoxicity and mutagenic activity in a mammalian in vitro cell system (Table III
). Concentrations of 500–5000 µg/ml ISIS 2302 were assessed in the presence and absence of S9 mix. Cytotoxicity was evident at the higher concentrations as a decrease in cell growth. Relative suspension growth from duplicate cultures decreased to 78% in response to 5000 µg/ml ISIS 2302. No cytotoxicity was observed in the subsequent mutagenesis experiments without S9. Despite cytotoxicity, the average cloning efficiency relative to the control cultures appeared to be unaffected by ISIS 2302 (Table III).
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There was no effect of ISIS 2032 on mutational frequency (MF) using standard experimental conditions in the presence or absence of S9 mix. The range of MF based on total TFTr colonies in vehicle control cultures ranged from 26x106 to 37x106 cells. However, a significant increase in mutation frequency (P < 0.01) was induced for the positive control compounds; ethylmethane sulfonate (EMS), methylmethane sulfonate (MMS) and 3-methylcholanthrene (MCA). MMS and MCA also increased the frequency of both small and large colonies, demonstrating that the test system was sensitive to known genotoxicants.
Cellular uptake and exposure in L5178Y mouse lymphoma cells was assessed in independent cultures. Analysis of cell extracts by CGE demonstrated that the concentrations of oligodeoxynucleotide and metabolites were dependent on initial medium concentration (Table I). The presence of S9 fraction had little or no effect on uptake. Immunohistochemical staining demonstrated intracellular localization of the oligodeoxynucleotide (data not shown).
Bone marrow mouse micronucleus assay
In the definitive assay one mouse died at 500 mg/kg and two died at 1000 mg/kg, demonstrating that the animals were sufficiently challenged and that the selected doses in this assay were in fact toxic. There were slight dose-related reductions in the PCE/RBC ratio at 48 and 72 h, however, these were not statistically significant between dose levels or groups (Table IV). No statistically significant increases in micronucleated PCEs were found using the Cochran–Armitage test for trend in binomial proportions or by the normal test for equality of binomial proportion were found in either gender at any time point in comparison with their respective controls (P 
0.05) (Kastenbaum and Bowman, 1970). The percentage of micronucleated PCEs in male mice treated with 250 mg/kg cyclophosphamide increased by 22-fold at the 48 h harvest and was statistically significant (Student's t-test). Control animal data were within the historical range for the testing facility (SRI, International, Menlo Park, CA) and, based on a total assessment of all study data, ISIS 2302 was clearly negative for this end-point.
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Exposure to oligodeoxynucleotide in the mouse micronucleus test was assessed by measuring plasma and bone marrow concentrations of ISIS 2302 and its metabolites in a separate experiment. Plasma concentrations 4 h post-dosing at 250 and 1000 mg/kg were 8.6 ± 4.8 and 35 ± 7.6 µg/ml, respectively (Table V
). Oligodeoxynucleotide metabolites 13–19 nt in length, corresponding to 20mer shortening by 1–7 nt, were measured in plasma. Plasma concentrations at 24 h were <5% of the concentration assessed at 4 h and were below the limit of quantitation in plasma for the 250 mg/kg dose at the 48 and 72 h time points (Table V).
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ISIS 2302 was distributed to bone marrow and exposure was more sustained relative to plasma concentrations. While bone marrow contained high concentrations of oligodeoxynucleotide at the 4 h time point, concentrations peaked 24 h after treatment and remained elevated throughout the duration of the study (72 h) (Table V
). The increased concentration of oligodeoxynucleotide in bone marrow at 24 h with a concurrent decrease in plasma concentration is indicative of tissue uptake. The concentration of oligodeoxynucleotide in bone marrow also increased as dose increased between 250 and 1000 mg/kg, but this increase was less than dose proportional. Bone marrow concentrations of ISIS 2302 at 24 h were 8.4 ± 3.4 and 17.6 ± 3.3 µg/g for the 250 and 1000 mg/kg dose levels, respectively. There was also significant exposure to metabolites in bone marrow. When metabolites were included in the calculation of concentration, bone marrow was exposed to total oligodeoxynucleotide concentrations of 18.6 and 43.6 µg/g in the 250 and 1000 mg/kg groups, respectively. Concentrations of parent oligodeoxynucleotide at 72 h remained within 50% of that determined at 24 h. A decrease in the percent of intact oligodeoxynucleotide from 44–50% at the 4 h time point to 33–37% at 72 h is consistent with slow biotransformation, presumably by ubiquitous exonucleases. Immunohistochemical staining of bone marrow for ISIS 2302 demonstrated an increase in staining 4 h after a dose of 1000 mg/kg relative to control bone marrow (data not shown). Staining in bone marrow from treated animals is consistent with cellular uptake. Concentrations of oligodeoxynucleotide in bone marrow are near the limit of detection by immunohistochemical staining and specific staining for oligodeoxynucleotide was difficult to distinguish at the lower dose. However, an apparent increase in staining was observed at the 250 mg/kg dose level and the intensity appeared to decrease with time. Under the conditions of this assay, bone marrow cells were exposed to significant concentrations of oligodeoxynucleotide for sufficient time and at sufficient concentrations to evaluate the risk of clastogenicity. Taken together with toxicity data, the negative results of the mouse bone marrow micronucleus assay cannot be attributed to inadequate exposure.
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Discussion |
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The potential therapeutic activity of oligodeoxynucleotides as antisense therapeutic agents is growing, with at least six compounds in late stage development. As such, it is important to understand the risk of genotoxicity of these compounds. ISIS 2302, like the other members of this class, showed no evidence of genotoxicity. The absence of genotoxicity may raise questions about exposure and the ability of the test cells and tissues to take up oligodeoxynucleotide. Therefore, supporting analytical measurements demonstrating intracellular localization of ISIS 2302 were performed. Clearly the evidence of toxicity observed in the test systems supports the concept that adequate exposure was achieved in the present series of assays.
ISIS 2302 is a phosphorothioate oligodeoxynucleotide that is composed of nucleotides derived from salmon sperm that are oligomerized in a specific sequence using standard DNA synthetic chemistry. The only difference between endogenous DNA and this compound is the substitution of sulfur for one of the non-bridging oxygens of the phosphodiester backbone. The sulfur substitution imparts nuclease resistance and increases tissue and plasma half-lives that are desirable for pharmacological efficacy. The pharmacological effects of antisense phosphorothioate oligodeoxynucleotides are the result of post-transcriptional binding to mRNA. Post-transcriptional effects should not alter the genome. The negative results in the genotoxicity studies presented herein support that hypothesis.
The inability of phosphorothioate oligodeoxynucleotides to interact with structured or double-stranded DNA results in part from differences in hybridization affinities between phosphodiester and phosphorothioate oligonucleotides. The affinity of a phosphorothioate oligonucleotide for its target is much less than the affinity of an endogenous DNA strand for its complement. Therefore, it is thermodynamically unfavorable for phosphorothioate oligodeoxynucleotides to compete for hybridization with double-stranded endogenous DNA (Freier, 1993; Lesnik et al., 1993; Lesnik and Freier, 1995). A similar argument can be made for the failure of phosphorothioate oligodeoxynucleotides to bind phosphodiester oligonucleotides with secondary structure. This latter factor limits target sites for phosphorothioate oligodeoxynucleotides in both genomic DNA and structured mRNA as well. The fact that antisense compounds like ISIS 2302 are relatively small and single-stranded also makes it unlikely that these compounds participate in homologous recombination. Characteristic of classic genetic recombination is the concept of branch migration (Holiday, 1964; Meselson and Radding, 1975), which involves strand breakage followed by the crossover and exchange of homologous sequences. Based on what is known about hybridization thermodynamics and limited physical size, there should be little if any chance for phosphorothioate oligodeoxynucleotides to participate in this type of event. The negative results obtained in the in vivo bone marrow micronucleus study and in vitro chromosomal aberration study in the present experiments support this conclusion.
The data in this report show that the absence of genotoxicity associated with ISIS 2302 in these assays cannot be ascribed to a failure of test cell exposure. For all of the assays reported, significant intracellular concentrations of parent oligodeoxynucleotide and chain-shortened oligodeoxynucleotide metabolites were achieved. The presence of metabolites is further evidence of cell uptake and ensures that genotoxicity of metabolites was evaluated. While these studies measured only the oligodeoxynucleotide metabolites, for each oligodeoxynucleotide metabolite formed there is one or more mononucleotide metabolite liberated. These mononucleotides can be catabolized, excreted or may enter endogenous pools. The monothiophosphate group of liberated mononucleotides may be removed by phosphatases, oxidized to phosphate or remain intact. In vivo studies with a phosphorothioate oligodeoxynucleotide labeled with 14C at the C-2 position of thymidine demonstrate that the radiolabel is excreted in the form of 14CO2 (Cossum et al., 1993, 1994). These data suggest that the thymidine liberated from the oligodeoxynucleotide was catabolized along the same pathways to the same end products as endogenous thymidine. In a similar fashion, the majority of radioactivity from an oligodeoxynucleotide labeled with 35S in the thioate linkage is excreted in urine and is associated with low molecular weight components (Agrawal et al., 1991; J.Leeds, unpublished observations). Together these results indicate that the majority of mononucleotides are catabolized or excreted. Should mononucleotide metabolites from ISIS 2302 enter the endogenous nucleotide pools, they would likely have unmodified nucleoside and ribose constituents. Incorporation of normal mononucleosides derived from antisense compounds would not be expected to result in mutations by mismatching during DNA replication.
One possible mechanism by which antisense compounds could produce genotoxicity is through alterations of endogenous nucleotide pools. There are reports that genotoxicity can result from alterations in the nucleotide pools (Bradley and Sharkey, 1978; Anderson et al., 1981). DNA precursor pool imbalances may induce mutation, recombination and chromosome aberrations and inhibit DNA repair in cultured cells and can modulate the genotoxicity of certain DNA-damaging agents (Kunz et al., 1994). However, the concentrations of nucleotides or nucleosides required to produce mutations in these studies exceed any that could be attained in vivo, even at the high doses used in toxicology studies. Furthermore, such a mechanism would have been detected in the present battery of tests performed for ISIS 2302.
In the current battery of tests the mouse micronucleus data are important as they allow consideration of in vivo metabolism, pharmacokinetics and DNA repair. Although intracellular metabolism of oligodeoxynucleotides within target cells in vitro was demonstrated, the data also suggest that exogenous metabolic activation systems typically used for genotoxicity assessments of small molecules are unlikely to be important. Unmistakably for this class of antisense therapeutics, bone marrow is a site of uptake and persistence. Furthermore, the high cellular proliferation rate of bone marrow and its sensitivity to micronucleated cell formation in response to known nucleotide perturbations make this tissue a predictor for genotoxicity through this mechanism. With this understood, ISIS 2302 clearly failed to cause increases in the frequency of micronucleated cells at doses producing systemic toxicity that are far in excess of those judged therapeutically beneficial.
The data presented in this paper demonstrate that ISIS 2302 is non-genotoxic in standard in vitro and in vivo tests. The absence of genotoxicity on the basis of the presented data could not be ascribed to a lack of target cell sensitivity or exposure. These negative findings for genotoxicity are consistent with what is currently known about the chemistry and mechanism of action of this novel new class of therapeutics.
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Notes |
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1 Present address: Sierra Biomedical, 10326 Roselle Street, San Diego, CA, USA
2 Present address: Transkaryotic Therapies Inc., 195 Albany Street, Cambridge, MA, USA
3 Present address: Midwest Research Institute, 1470 Treeland Boulevard South East, Palm Bay, FL, USA
4 Present address: SRI International, 333 Ravenwood Avenue, Menlo Park, CA 94025, USA
5 To whom correspondence should be addressed. Tel: +1 760 603 3813; Fax: +1 760 603 3862; Email: shenry{at}isisph.com
6 These authors contributed equally to the data collection and presentation in this manuscript and are to be considered joint first authors
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References |
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Received on December 13, 2001; accepted on January 1, 2002.
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