Acute, Multiple-Dose, and Genetic Toxicology of AR177, an Anti-HIV Oligonucleotide

Thomas L. Wallace*,1, Christina Gamba-Vitalo{dagger},2, Ken S. Loveday{dagger},3 and Paul A. Cossum*

* Aronex Pharmaceuticals, Inc., The Woodlands, Texas 77381; {dagger} Genzyme TSI Mason, Worcester, Massachusetts

Received September 25, 1998; accepted June 3, 1999


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
AR177 (ZintevirTM) is a 17-mer oligonucleotide that has been shown to have anti-HIV activity and to be a potent HIV-1 integrase inhibitor in vitro, and is among the first oligonucleotides to enter human clinical trials. Acute and multiple-dose intravenous toxicity studies were performed in mice, and genetic toxicity studies were performed in vitro and in vivo in order to determine the toxicity profile of AR177. The acute toxicity study in mice showed that AR177 had an LD50 of >=1.5 g/kg body weight. The multiple-dose toxicity study in mice showed that AR177 caused male-specific mortality, and changes in serum chemistry, hematology, and histology at doses of 250 and 600 mg/kg. Clinical chemistry findings included changes in liver function, and decreased erythrocyte values at 250 and 600 mg/kg. Histopathologic findings included vacuolization of reticuloendothelial cells in phagocytic cells in lymphoid tissue, liver, lungs, heart and uterus, and extramedullary hematopoeisis in the spleen. Renal toxicity was exhibited as nephropathy and tubular necrosis in the two high-dose groups of males. A no-effect dose was not established. AR177 did not exhibit genetic toxicity in any of three mutagenic assays. In combination with previously reported toxicity studies of AR177 in monkeys, this study showed that the toxicity of AR177 is species specific.

Key Words: Zintevir; HIV; oligonucleotide; mice; toxicity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oligonucleotides, long used as probes in molecular biology, are now considered potential therapeutics for the treatment of a variety of human diseases. Virtually any type of human clinical disorder can theoretically be treated with oligonucleotides, given knowledge of a molecular target that is involved in either the cause or sequelae of the disorder. The clinical applications of oligonucleotide drugs have been quite varied to date. Cancer, viral disorders including HIV (human immunodeficiency virus) infection, inflammation, genital warts, and restenosis are some of the clinical disorders for which oligonucleotides are now being used in humans (Zon, 1995Go). Fomivirsen (ISIS 2922), an antisense oligonucleotide used to treat cytomegalovirus (CMV) retinitis in AIDS patients, is the first oligonucleotide to receive marketing approval by the U.S. Food and Drug Administration.

One of the important issues of oligonucleotides, as with any new class of drugs, is their toxicity profile in animals and humans. Several questions have arisen. One question is whether toxicity studies in animals will be predictive of the toxicity profile in humans. Another question is whether oligonucleotides with different sequences have similar toxicity profiles. A third question is whether backbone modifications of oligonucleotides contribute to the toxicity of the compounds.

AR177 (ZintevirTM; T30177; 5'-GTGGTGGGTGGGTGGGT-3') is a 17-mer oligonucleotide that is currently in repeat-dose phase I clinical trials for the treatment of HIV infection. This oligonucleotide is composed entirely of deoxyguanosines and thymidines, which impart a compact tetrad structure believed to be responsible for its potent inhibition of HIV-1 integrase activity (Ojwang et al., 1995Go; Rando et al., 1995Go), unusual biochemical stability (Bishop et al., 1996Go), low toxicity in primates (Wallace et al., 1996aGo, 1996bGo), and long half-life in rats (Wallace et al., 1997Go). AR177 is the most potent HIV-1 integrase inhibitor that has been reported (Mazumder et al., 1996Go) and is the first integrase inhibitor to enter human clinical trials for the treatment of HIV infection.

As part of the preclinical assessment of AR177, acute and multiple-dose intravenous toxicity studies were performed in mice, and genetic toxicity studies were performed in vitro and in mice. These studies have revealed that the toxicity profile in mice is different than that previously reported for AR177 in monkeys, and for other oligonucleotides in mice. They have also revealed that AR177 does not have any toxicity in three common mutagenic assays. These data contribute to the growing body of studies that indicate that the toxicity profile of oligonucleotides can be both sequence and species specific.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
AR177 was synthesized at Biosearch, a division of PerSeptive Biosystems, on a Milligen 8800 oligonucleotide synthesizer (Millipore, Bedford, MA), and vialed at 25 mg/ml in phosphate-buffered saline (PBS). AR177 has a molecular weight of 5,793 daltons, and is a fully neutralized sodium salt. The structure of AR177 was characterized by phosphorus and proton NMR, sequencing, base composition, laser desorption mass spectrometry, anion exchange HPLC, and polyacrylamide gel electrophoresis. The AR177 was approximately 94% pure according to HPLC and electrophoretic analysis. All analyses were consistent with the proposed structure.

Acute and Fourteen-Day Multiple-Dose Intravenous Toxicity Studies
Animals.
Mice were purchased from Charles River Laboratories (Wilmington, MA) and were quarantined for 2–3 weeks before dosing. The animals were randomized into groups using a computer-generated randomization program. At randomization, the weight variation was within ± 20% of the mean weight within each sex. The mice were housed in stainless steel cages and fed LabDietTM Certified Rodent Diet 5002 (PMI Feeds, Inc.). Food and water were available ad libitum. The mice were housed in a room with a temperature of 70 ± 3°F, a relative humidity of 31 to 62%, and a 12-h light/dark cycle. The room had a minimum of 10 fresh air changes per hour. All husbandry conditions were performed according to the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86–23, Revised 1985).

Dosing.
In the acute toxicity study, mice were injected intravenously through the tail vein with a total dose of 100, 500, or 1,500 mg/kg/mouse of AR177 in PBS (Table 1Go). The top dose of 1500 mg/kg was the maximum possible, given the concentration of the stock solution (25 mg/ml) and the highest allowable dosing volume (60 ml/kg/day). A control group received PBS only. The dose was split into two injections, administered as two 30 ml/kg injections 5 h apart. There were five male and five female mice per dose group. In the fourteen-day multiple-dose toxicity study, mice were injected intravenously twice per day for 14 days through the tail vein with PBS (Group 1), or AR177 in PBS at a total dose of 50 (Group 2), 250 (Group 3), or 600 (Group 4) mg/kg/mouse/day. See Table 2Go. Because of unanticipated deaths and moribundity in Group 3 and 4 males, the dose level for Group 3 animals was decreased from 250 mg/kg/day to 100 mg/kg/day on day 9 and the dose level for Group 4 animals was decreased from 600 mg/kg/day to 400 mg/kg/day starting on day 5 of study. A control group received PBS only. There were six male and six female mice per dose group. The dose was split into two injections, which were administered 5 h apart. The total dose volume was maintained at 60 ml/kg, administered as 30 ml/kg twice a day approximately 5 h apart. Animals were observed until day 15, after which they were euthanized and subjected to a gross necropsy.


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TABLE 1 Dosing Table for the Acute Intravenous Toxicity Study of AR177 in Mice
 

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TABLE 2 Dosing Table for the 14-day Intravenous Toxicity Study of AR177 in Mice
 
Assessment of toxicity.
In the acute toxicity study, animals were observed until day 15, after which they were euthanized and subjected to a gross necropsy. Evidence of a toxic effect was assessed by clinical observations, body weights, gross necropsy findings, and organ weights. Clinical observations included the skin and hair, eyes and mucous membranes; respiratory system, circulatory system, central nervous system, somatomotor activity; and the occurrence of tremors, convulsions, salivation, diarrhea, or lethargy. Body weights were recorded prior to treatment, on days 7 and 14, and at euthanasia (day 15) or death. The necropsy included examination of the external surface of the body, orifices, and the cranial, thoracic, and abdominal cavities and their contents. Forty-eight organs and tissues were examined at necropsy. Organ weights of the adrenals, brain, kidney, liver, pituitary, testes, and thyroid gland were recorded. In the fourteen-day multiple-dose toxicity study, toxicity was assessed by daily clinical observations, weekly body weights, food consumption, and clinical pathology parameters. Moribund mice were sacrificed by CO2 inhalation. Blood samples were obtained by retro-orbital sinus puncture after anesthetization with CO2 for the clinical pathology evaluations. Half of surviving mice were bled for hematology parameters and half of the surviving mice were bled for clinical chemistry parameters prior to sacrifice. Hematology parameters were measured using a Sysmex TOA E-2500 hematology analyzer. The hematology parameters included red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, absolute and relative nucleated red blood cell count, anisocytosis, poikilocytosis, hypochromasia, polychromasia, hypochromasia, white blood cell count, absolute and relative polymorphonuclear neutrophil count, absolute and relative band count, absolute and relative lymphocyte count, absolute and relative monocyte count, absolute and relative eosinophil count, absolute and relative basophil count, and platelets. Clinical chemistry parameters were measured using a Boehringer Mannheim Hitachi 717 chemistry analyzer. The clinical chemistry parameters included glucose, blood urea nitrogen, creatinine, total protein, albumin, globulin, total bilirubin, cholesterol, triglycerides, calcium, phosphorus, sodium, potassium, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and gamma glutamyltransferase. All surviving animals were euthanized on day 15 and subjected to a comprehensive necropsy. Weights were taken for the same organs as in the acute toxicity study. A histopathologic assessment was made of 39 sectioned and hematoxylin- and eosin-stained tissues by a board-certified veterinary pathologist. These included tissues from the cardiovascular, digestive, endocrine, reproductive, integument, hematopoietic, nervous, musculoskeletal, and urinary systems.

Statistical analysis.
The distribution of the data was assessed using the Bartlett Test for homogeneity of variance. If this result indicated a parametric distribution (p > 0.05), then subsequent statistical evaluations were performed using one-way analysis of variance (ANOVA). A significant overall ANOVA of p <=0.05 was followed by a Dunnett's t Test to reveal differences between control and each treatment group. If the Bartlett Test yielded a significant probability (p <=0.05), then the data was considered nonparametric and a Kruskal-Wallis Test for independent groups was performed. With overall significance established, a Dunn's t Test was used to determine differences between control and each treatment group. A 95% confidence level (p <= 0.05) was the criteria for statistical significance.

Genetic Toxicology Studies
Ames/Salmonella. mutagenesis assay.
The purpose of this study was to investigate the ability of AR177 to induce mutations in Salmonella typhimurium using an in vitro mutagenesis assay. AR177 was tested for its ability to induce mutations in five strains of Salmonella typhimurium, TA98, TA100, TA102, TA1535, and TA1537, in the absence and presence of a metabolic activation system derived from rat liver S9 fraction. AR177 was tested in a range of 0.015 to 5.0 mg/plate. There were three plates tested per strain of Salmonella per experiment, and there were two experiments. Positive control agents were 2-aminoanthracene for activated TA98, TA100, TA1535, and TA 1537; 1,8-dihyroxyanthraquinone for activated TA102; 9-aminoacridine for nonactivated TA1537; sodium azide for nonactivated TA100 and TA1535; 2-nitrofluorene for nonactivated TA98; and cumene hydroperoxide for nonactivated TA102. PBS was the negative control. The Ames test was performed according to Gatehouse et al. (1994).

In vitro CHO/HGPRT mammalian cell mutagenesis assay.
The purpose of this study was to assess the ability of AR177 to induce mutations at the HGPRT locus in cultured mammalian cells (Chinese hamster ovary line). AR177 was evaluated in the presence and absence of a metabolic activation system derived from rat liver (S9). Ethylmethanesulfonate was the positive control for the nonactivated assay and dimethylbenzathracene was the positive control for the activated assay. PBS was the negative control. The AR177 concentrations for the nonactivated assay ranged from 15 to 5009 µg/ml, and the AR177 concentrations for the activated assay ranged from 487 to 4868 µg/ml. The mutation frequency was calculated as the total number of mutants divided by the (total number of cells plated x the cloning efficiency). This test was performed according to the procedure of Galloway et al. (1994).

In vivo mouse micronucleus assay.
AR177 was evaluated for its ability to induce micronuclei in the bone marrow of B6C3Fl mice. Cyclophosphamide was the positive control and 0.9% saline was the negative control. Groups of five male and five female B6C3Fl mice were dosed on 3 consecutive days via intravenous injection and sacrificed 24 h after the last dosing. Mice were dosed with AR177 at 106, 353, and 1058 mg drug/kg. Bone marrow cells from animals were analyzed for the number of polychromatic erythrocytes (PCEs) that contained at least one micronucleus. A minimum of 2000 PCEs was analyzed for the vehicle controls and for mice treated with the AR177. A minimum of 1000 PCEs was analyzed for each positive control animal. The PCE fraction was determined by counting a minimum of 200 erythrocytes (PCEs plus normochromatic erythrocytes [NCEs]). This test was performed according to Hayashi et al. (1994).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Acute Intravenous Toxicity Study of AR177 in Mice
Mice were injected intravenously with a single dose of AR177 at 100 (Group 2), 500 (Group 3), or 1500 (Group 4) mg/kg (Table 1Go). Three females and one male in Group 4 died following the single dose. Two of the Group 4 females died on day 1. One Group 4 male died on day 2 and one of the Group 4 females died on day 5 of study. Therefore, the LD50 was >=1500 mg/kg. Clinical observations that were attributed to the administration of AR177 were lethargy, urine stains, coldness to the touch, prostration, tremors and swelling of the face. There were no treatment-related effects on body weight for males or females (data not shown). There were no dose dependent changes in organ weights. There were no treatment-related gross necropsy findings for either scheduled euthanasia or early death animals in the study.

Fourteen-Day Repeat Dose Intravenous Toxicity Study of AR177 in Mice
Mice were injected intravenously with repeated doses of AR177 (Table 2Go). Two Group 3 (250/100 mg/kg) males were found dead on days 7 and 12. One Group 4 (600/400 mg/kg) male was found dead on day 6, and two Group 4 males were euthanized due to moribundity on day 6. All females survived until scheduled necropsy. These animals exhibited one or more of the following clinical observations: thin appearance, hunched posture, ruffled coat, urine stains, labored breathing, lethargy, tremors, and coldness. Ruffled coat was noted in at least half of the males assigned to Groups 2, 3, and 4 and two Group 4 females. There was no significant effect of AR177 on body weight by the day 14 time point (data not shown). Decreased food consumption was evident in Group 3 and 4 males during week 1 (data not shown).

AR177-related findings included increased alanine aminotransferase levels (Fig. 1Go), increased aspartate aminotransferase levels (Fig. 2Go), decreased albumin values, protein values, and albumin/globulin ratios (Fig. 3Go), decreased red blood cell number (Fig. 4Go), hemoglobin (Fig. 5Go), hematocrit (Fig. 6Go), mean corpuscular hemoglobin (Fig. 7Go), and mean corpuscular hemoglobin concentration (data not shown). All other clinical chemistry and hematology values were normal. Decreases in red blood cell number and hematocrit caused by oligonucleotides have also been reported by Henry et al. (1997e).



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FIG. 1. Effect of multiple intravenous doses of AR177 on serum alanine aminotransferase levels in mice. Values are the mean of 2–4 mice. Where n >= 3, a standard deviation is shown. Dose group means significantly different from the placebo mean are denoted by an asterisk.

 


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FIG. 2. Effect of multiple intravenous doses of AR177 on serum aspartate aminotransferase levels in mice. Values are the mean of 2–4 mice. Where n >= 3, a standard deviation is shown.

 


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FIG. 3. Effect of multiple intravenous doses of AR177 on serum albumin/globulin ratio in mice. Values are the mean ± SD (n = 3). Dose group means significantly different from the placebo mean are denoted by an asterisk.

 


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FIG. 4. Effect of multiple intravenous doses of AR177 on red blood cell concentration in mice. Values represent 1–3 mice. Where n = 3, a standard deviation is shown.

 


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FIG. 5. Effect of multiple intravenous doses of AR177 on hemoglobin concentration in mice. Values represent 1–3 mice. Where n = 3, a standard deviation is shown.

 


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FIG. 6. Effect of multiple intravenous doses of AR177 on hematocrit in mice. Values represent 1–3 mice. Where n = 3, a standard deviation is shown.

 


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FIG. 7. Effect of multiple intravenous doses of AR177 on mean corpuscular hemoglobin concentration in mice. Values represent 1–3 mice. Where the n = 3, a standard deviation is shown.

 
Histopathologic analysis of protocol-specified tissues revealed AR177-related findings in Group 2 through Group 4 animals (Tables 3 and 4GoGo). These microscopic findings consisted of enlarged vacuolated reticuloendothelial cells in mandibular and mesenteric lymph nodes, Kupffer cells in the liver, septal cells lining alveolar walls in the lung, and mononuclear cells of the spleen. Vacuolated histiocytes were observed in the interstitial connective tissue of the myocardium of one-half of the Group 4 animals and in the interstitial connective tissue of the uterus of all Group 4 females. Vacuolization of proximal tubular epithelial cells of the outer cortex was noted in the kidneys of the Group 4 females and nephropathy and tubular epithelial necrosis was noted in several Group 3 and 4 male mice. Extramedullary hematopoeisis in the spleen was slightly increased in Group 4 males and females. These microscopic observations in the liver and spleen correlated well with the increased liver weight values in Group 2 through Group 4 males and females (Fig. 8Go) and the enlarged spleens noted in Group 4 animals at necropsy. Based upon the results in this study, a no-effect level (NOEL) was not established, and based on mortality, males appeared more sensitive than females to the AR177.


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TABLE 3 Histopathological Changes in Male Mice After Multiple Intravenous Doses of AR177
 

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TABLE 4 Histopathological Changes in Female Mice After Multiple Intravenous Doses of AR177
 


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FIG. 8. Effect of multiple intravenous doses of AR177 on liver as a percentage of body weight in mice. Values are the mean ± SD (n = 3–6).

 
Ames/Salmonella Mutagenesis Assay
AR177 did not induce mutations in any of the strains tested, either in the absence or presence of S9, in any of the six plates tested in the two experiments (data not shown). All positive controls were active against all appropriate Salmonella strains. AR177 was not toxic to any of the five Salmonella strains.

In Vitro CHO/HGPRT Mammalian Cell Mutagenesis Assay
The mutation frequency was based on mutants per 1x106 clonable cells. In the nonactivated assay, the mean mutation frequency was 4.3 for the saline negative control; 393 for the ethylmethanesulfonate positive control; 4.7 for the 501 µg/ml concentration of AR177; 5.3 for the 1503 µg/ml concentration of AR177; and 1.4 for the 5009 µg/ml concentration of AR177. In the activated assay, the mean mutation frequency was 1.7 for the saline negative control; 189 for the dimethylbenzathracene positive control; 6.6 for the 487 µg/ml concentration of AR177; 7.4 for the 1460 µg/ml concentration of AR177; and 4.8 for the 4868 µg/ml concentration of AR177. Similar results were obtained in a second experiment. Although the AR177 mutation values were higher than the negative control in the activated assay, they were not indicative of a mutagenic response. AR177 was not cytotoxic at the concentrations tested. Thus, AR177 was nonmutagenic to CHO cells in this study.

In vivo Mouse Micronucleus Assay
The micronucleated PCE means ± SE per 1000 PCE in male mice (n = 5) were 2.0 ± 0.5 for the saline control; 20.2 ± 2.1 for the cyclophosphamide control; 2.1 ± 0.3 for the 106 mg/kg dose; 2.7 ± 0.4 for the 353 mg/kg dose; and 2.0 ± 0.4 for the 1058 mg/kg dose. The micronucleated PCE means ± SE per 1000 PCE in female mice (n = 5) were 2.1 ± 0.2 for the saline control; 25.4 ± 1.7 for the cyclophosphamide control; 1.7 ± 0.3 for the 106 mg/kg dose; 1.8 ± 0.6 for the 353 mg/kg dose; and 1.7 ± 0.3 for the 1058 mg/kg dose. Thus, AR177 did not induce an increase in the frequency of micronucleated polychromatic erythrocytes relative to the vehicle control in either male or female mouse bone marrow. No decrease in the polychromatic erythrocyte fraction was found in AR177-treated male or female animals compared to the vehicle controls, indicating that no cytotoxicity was caused by AR177.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oligonucleotides have been examined in a number of toxicity studies in mice and monkeys. In mice, Sarmiento et al. (1994) reported that a rel A antisense phosphorothioate oligonucleotide caused mortality in 7/12 mice at a dose of 150 mg/kg and in 2/12 mice at a dose of 100 mg/kg after intraperitoneal dosing three times per week for 2 weeks. Clinical chemistry findings included severe thrombocytopenia, and elevated liver enzymes. Histopathologic findings included severe cortical toxicity in the kidney, thymus, and adrenal at the 150 mg/kg dose. Henry et al. (1997d) reported that ISIS phosphorothioate oligonucleotide 2302, directed against human ICAM-1 mRNA, did not cause mortality when it was administered intravenously every other day for 27 days at doses up to 100 mg/kg. Immune stimulation and hepatic toxicity were seen at the 100 mg/kg dose. Monteith et al. (1997) have reported that immune stimulation is a class effect of phosphorothioate oligodeoxynucleotides. Henry et al. (1997e) reported that ISIS phosphorothioate oligonucleotide 3082, directed against mouse ICAM-1, did not cause mortality when it was administered intravenously every other day for 14 days at 50 mg/kg. Splenomegaly, hepatic toxicity, and decreases in triglycerides, cholesterol, and platelets were seen. Five structurally related oligonucleotides had a similar toxicity profile. In the present study, AR177 had an LD50 of greater than 1.5 g/kg body weight when it was given as an acute dose. When AR177 was given intravenously once per day for 14 days, it caused mortality at doses of 250 and 600 mg/kg in male but not female mice. Clinical chemistry findings included changes in liver function, and decreased erythrocyte values at 250 and 600 mg/kg. Histopathologic findings included vacuolization of reticuloendothelial cells in the phagocytic cells in lymphoid tissue, liver, lungs, heart, and uterus. Renal toxicity was exhibited as nephropathy and tubular necrosis in the two high-dose groups of males. It may be concluded that the toxicity profile of oligonucleotides in these aforementioned studies appears to be somewhat different, although there were significant differences in the frequency, route of administration, and dose in these studies.

In monkeys, there appear to be two types of toxicity that are of particular concern in primates: hemodynamic toxicity, manifested as alterations in blood pressure, and hematologic toxicity, manifested as alterations in white blood cells and increases in clotting times. Cornish et al. (1993) reported that a phosphorothioate oligonucleotide designated OL(1)p53 caused death following a drop in blood pressure in a rhesus monkey when it was given by intravenous injection of 8.3 mg/kg over 4 min, but no mortality when the oligonucleotide was administered by slow infusion. A human study using this oligonucleotide showed little toxicity following intravenous infusion at a much reduced rate of infusion of up to 0.25 mg/kg/hr for 10 days (5.28 grams of oligonucleotide) (Bayever et al., 1993Go; Bishop et al., 1996Go). Galbraith et al. (1994) reported that GEM91, a phosphorothioate oligonucleotide, caused lymphopenia, neutropenia, and a profound drop in blood pressure in monkeys when it was administered intravenously at either 80 mg/kg over 2 h or at 20 mg/kg over 10 min. Significant complement activation was associated with the blood pressure drop. Henry et al. (1997a) reported that ISIS oligonucleotide 2302 did not cause any deaths in monkeys when it was administered intravenously at doses of up to 50 mg/kg every other day for 28 days. Dose-dependent prolongation of clotting times, subcutaneous hemorrhages, and vacuolation of the renal tubular cells were seen. Wallace et al. (1996a) reported that AR177, a partial phosphorothioate, did not cause any mortality or changes in blood pressure, ECG, clinical chemistry, or hematologic parameters after doses of 5, 20, or 50 mg AR177/kg given as a 10-min intravenous infusion to monkeys. There was minimal, dose-dependent complement activation increase in the level of the complement split product Bb and CH50, but there was a significant dose-dependent and reversible inhibition of coagulation with the 20 and 50 mg/kg doses that lasted up to several hours postinfusion. The time course of the inhibition of coagulation closely matched the plasma levels of AR177. In a repeat-dose toxicity study, AR177 was administered at doses up to 40 mg/kg every other day for 23 days with no evidence of histopathology (Wallace et al., 1996bGo).

Both the hemodynamic and hematologic toxicities in primates have been linked to complement activation. Henry et al. (1997b) have reported that the mechanism by which the ISIS phosphorothioate oligonucleotide 2302 causes complement activation is through the alternative complement pathway, possibly by a direct interaction between the oligonucleotide and Factor H. However, it is clear that some oligonucleotides cause significant hemodynamic toxicity, whereas others do not. The degree of hemodynamic alterations is dependent upon the degree of thiation, with total phosphorothioates causing more toxicity than partial phosphorothioates. This is supported by the work of Henry et al. (1997c), which showed that inhibition of coagulation was increased with increasing thiation of ISIS oligonucleotide 2302. It has been reported that rodents do not exhibit the same hypotensive events that are seen in monkeys (Black et al., 1994Go), indicating that this effect is primate specific and is a necessary parameter to examine in monkeys before proceeding to human studies (Black et al., 1994Go). Studies have shown that the degree of hemodynamic and/or hematologic toxicity in primates is directly related to the plasma concentration of drug that is achieved (Henry et al., 1997eGo; Wallace et al., 1996bGo), suggesting that these toxicities can be minimized by slowing the time of intravenous drug infusion.

In conclusion, a toxicity profile in several animal species of an oligonucleotide will be important for understanding the potential toxicities that could be seen in humans and whether toxicity results in animals can be predictive of the toxicity profile in humans. The acute toxicity in mice, cardiovascular toxicity in monkeys, repeat-dose toxicity in mice, repeat-dose toxicity in monkeys, and genetic toxicity profiles of AR177 have now been published and will provide a basis on which to compare other oligonucleotides. The results indicate that the toxicity of AR177 is species specific, with renal necrosis and histologic vacuolization being prominent in several organs in mice. Comparable pathology was not seen with AR177 in cynomolgus monkeys, although lower doses were administered. No toxicity has been seen in humans at doses up to 3 mg/kg given every day for 14 days (unpublished data).


    ACKNOWLEDGMENTS
 
This work was supported in part by Phase I Small Business Innovation Grant 1 R43 AI38788–01 from the National Institute of Allergy and Infectious Diseases (T. L. W.)

The authors thank Dr. Dennis Mulvey, Dr. Ira Goldknopf, Nancy Joyce, and David Walker for the characterization of AR177.


    NOTES
 
1 To whom correspondence should be addressed at Aronex Pharmaceuticals, Inc., 8707 Technology Forest Place, The Woodlands, TX 77381. Fax: (713) 367–1676. E-mail: twallace{at}aronex-pharm.com. Back

2 Present address: Antigenics L. L. C., New York, NY. Back

3 Present address: Creative Biomolecules, Hopkinton, MA. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bayever, E., Iversen, P. L., Bishop, M. R., Sharp, J. G., Tewary, H. K., Arneson, M. A., Pirruccello, S. J., Ruddon, R. W., Kessinger, A., Zon, G., and Armitage, J. O. (1993). Systemic administration of a phosphorothioate oligonucleotide with a sequence complementary to p53 for acute myelogenous leukemia and myelodysplastic syndrome: Initial results of a phase I trial. Antisense Res. Dev. 3, 383–390.[Medline]

Bishop, J. S., Guy-Caffey, J. K., Ojwang, J. O., Smith, S. R., Hogan, M. E., Cossum, P. A., Rando, R. F., and Chaudhary, N. (1996). Intramolecular G-quartet motifs confer nuclease resistance to a potent anti-HIV oligonucleotide. J. Biol. Chem. 271, 5698–5703.[Abstract/Free Full Text]

Black, L. E., Farrelly, J. G., Cavagnaro, J. A., Ahn, C. H., DeGeorge, J. J., Taylor, A. S., DeFelice, A. F., and Jordan, A. (1994). Regulatory considerations for oligonucleotide drugs: updated recommendations for pharmacology and toxicology studies. Antisense Res. Dev. 4, 299–301.[ISI][Medline]

Cornish, K. G., Iversen, P., Smith, L., Arneson, M., and Bayever, E. (1993). Cardiovascular effects of a phosphorothioate oligonucleotide with sequence antisense to p53 in the conscious rhesus monkey. Pharmacol. Commun. 3, 239–247.

Galbraith, W. M., Hobson, W. C., Giclas, P. C., Schechter, P. J., and Agrawal, S. (1994). Complement activation and hemodynamic changes following intravenous administration of phosphorothioate oligonucleotides in the monkey. Antisense Res. Dev. 4, 201–206.[ISI][Medline]

Galloway, S. M., Aardema, M. J., Ishidate M., Jr, Ivett, J. L., Kirkland, D. J.., Morita, T., Mosesso, P., and Sofuni, T. (1994). Report from working group on in vitro tests for chromosomal aberrations. Mutat. Res. 312, 241–261.[ISI][Medline]

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Henry, S. P., Bolte, H., Auletta, C., and Kornbrust, D. J. (1997a). Evaluation of the toxicity of ISIS 2302, a phosphorothioate oligonucleotide, in a four-week study in cynomolgus monkeys. Toxicology 120, 145–155.[ISI][Medline]

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