Animal safety and toxicology of simvastatin and related hydroxy-methylglutaryl-coenzyme A reductase inhibitors.

Simvastatin, a hydroxy-methylglutaryl-coenzyme A reductase inhibitor intended for use as a hypocholesterolemic agent, has undergone a thorough preclinical toxicology evaluation. This review describes preclinical toxicology findings associated with simvastatin administration in animals and provides the rationale for our conclusion that these changes are not indicative of potential human toxicity. Although it was not surprising to find that a potent inhibitor of this key biochemical pathway produces toxicity at high dosages in animals, none of the observed changes poses a significant risk to humans at clinical dosages. Many of the toxicities produced by high dosage levels of simvastatin in animals are directly related to the drug's biochemical mechanism of action and are the result of a profound, sustained inhibition of the target enzyme that is not anticipated at clinical dosages. Furthermore, several of the simvastatin-induced changes are species-specific responses to this agent and are not relevant to human risk assessment. Of the treatment-related changes reported for simvastatin, the development of cataracts in dogs has received considerable attention. The available data demonstrate a wide margin of safety in terms of dosage levels required to elicit this response as well as the plasma concentrations associated with the development of these ocular lesions. The data suggest that the development of lenticular opacities at clinical doses of simvastatin is highly improbable. Overall, simvastatin is highly improbable. Overall, simvastatin was well-tolerated by animals in preclinical toxicology studies, and no findings contraindicating its use in humans were identified.

Preclinical evaluation of lovastatin.

Administration of lovastatin to animals at high dosage levels produces a broad spectrum of toxicity. This toxicity is expected based on the critical nature of the target enzyme (HMG CoA reductase) and the magnitude of the dosage levels used. The information reviewed in this paper demonstrates that these adverse findings in animals do not predict significant risk in humans. The reason for this derives from the fact that all the available evidence suggests that the adverse effects observed are produced by an exaggeration of the desired biochemical effect of the drug at high dosage levels. The presence of clear and high no-effect doses for these toxic effects along with the fact that most of the changes observed are clearly mechanism-based (directly attributable to inhibition of mevalonate synthesis) indicate that it is unlikely that similar changes will be observed at the therapeutic dosage levels in humans. This hypothesis is supported by the extensive human safety experience described by Tobert in the following report.

Evaluation of the toxicity of ISIS 2302, a phosphorothioate oligonucleotide, in a four-week study in cynomolgus monkeys.

The toxicity of ISIS 2302, a phosphorothioate oligonucleotide with antisense activity against human ICAM-1 mRNA, was investigated in cynomolgus monkeys (young adult). The oligonucleotide was administered by slow bolus injection every other day for 28 days (14 doses) at dose levels of 0, 2, 10, and 50 mg/kg/injection. The basic group size consisted of three male and three female monkeys which were sacrificed 2 days after the last dose. An additional 2 monkeys/sex in the vehicle control and 50 mg/kg dose groups remained on study for a 28-day treatment-free period. No treatment-related deaths occurred during this study, however, one monkey in the 10 mg/kg dose group was markedly lethargic after the first dose. Other clinical observations included periocular swelling (> or = 10 mg/kg) on the first day of the study, and bruising in all dose groups throughout the study. Bruising was associated with a dose-dependent prolongation of clotting times, particularly activated partial thromboplastin times (APTT), that was transient in nature. Bruises occurred around site of intravenous dosing or blood collection, and were manifested as subcutaneous hemorrhages upon microscopic evaluation. There were no corresponding alterations in hematology parameters including RBC or platelet counts. Other treatment-related microscopic alterations noted were intracytoplasmic eosinophilic granules and vacuolation in proximal tubular epithelial cells at 10 and 50 mg/kg, with free RBC in renal proximal tubular lumens at 50 mg/kg. Serum chemistry parameters including BUN and creatinine levels were normal in all dose groups and there were no notable alterations in urinalysis parameters. Granules and vacuolations in kidneys were reversed following a 4-week treatment free period. In general, 10 and 50 mg/kg ISIS 2302 produced dose-dependent changes in clotting times and the kidney that were reversible, while 2 mg/kg ISIS 2302 produced no remarkable alterations.

Comparison of the toxicity profiles of ISIS 1082 and ISIS 2105, phosphorothioate oligonucleotides, following subacute intradermal administration in Sprague-Dawley rats.

The systemic toxicity of two phosphorothioate oligonucleotides specific for herpes simplex viruses (ISIS 1082) and human papiloma virus (ISIS 2105) were evaluated following repeated intradermal injections of vehicle control, 0.33, 2.17, or 21.7 mg/kg daily to Sprague-Dawley rats (10/sex/group) for 14 days. Animals were sacrificed 1 day after the last dose, except for a portion of the ISIS 1082-treated animals (5/sex/group) which were maintained for an additional 14-day recovery period. The profile of alterations noted for both compounds was very similar. Other than local signs of irritation at the site of injection, there were no clinical signs of toxicity or treatment-related mortality, but there was a slight decrease in body weight gain for the 21.7 mg/kg dose groups. Alterations in hematology parameters included dose-dependent thrombocytopenia and anemia. Alterations in serum chemistry parameters were suggestive of mild alterations in hepatic metabolism, with increases in liver transaminases and bilirubin, along with decreases in albumin and cholesterol. Both spleen and liver weights were significantly elevated in a dose-dependent fashion. Histopathological alterations noted in liver, kidney, lung, injection site skin, and spleen were characterized as perivascular and interstitial infiltrates of macrophages and monocytes. Additional microscopic alterations in the spleen included mild lymphoid hyperplasia (seen in lymph nodes as well), and extramedullary hematopoiesis. Treatment-related cytopenias were likely related to mild, focal hypocellularity in the bone marrow. Alterations in ISIS 1082-treated animals were only partially reversed following the 14-day treatment-free period. In conclusion, repeated intradermal administration of ISIS 1082 and ISIS 2105 produced a similar spectrum of toxicities, with liver, kidney, spleen, and bone marrow being identified as target tissues.

Comparison of 7 azo dyes and their azo reduction products in the rat and hamster hepatocyte primary culture/DNA-repair assays.

Pursuant to the characterization of species differences in the effects of chemical carcinogens, several studies have demonstrated that hamster hepatocytes are more effective than rat hepatocytes in mediating the metabolic activation of certain chemicals to their genotoxic (i.e., mutagenic) derivatives. In the present investigation, a comparison of the amount of DNA repair induced in rat and hamster hepatocytes by 7 azo dyes and 7 aromatic amine azo reduction products of the dyes was performed using the primary hepatocyte culture/DNA repair (HPC/DR) assay. Congo Red and its azo reduction product, benzidine, were more potent inducers of DNA repair in hamster than in rat hepatocytes, whereas Trypan Blue and its reduction product, o-tolidine, were equipotent in the 2 hepatocyte systems. Evans Blue, another o-tolidine-based dye, elicited a greater DNA-repair response in hamster hepatocytes. The absolute potency of these dyes, however, was much less than their reduction products. o-Aminoazotoluene was the most potent of the dyes tested, and its DNA repair-inducing activity was much greater than that of its azo reduction products, o-toluidine and 2,5-diaminotoluene. Ponceau SX, which is carcinogenic in hamsters, but not in rats, was inactive in both hepatocyte systems. Dimethylaminobenzeneazo-1-naphthalene and its 2-naphthalene congener, as well as the 1- and 2-naphthylamine azo reduction products of these dyes, were more potent in hamster than in rat hepatocytes. However, the DNA repair-inducing activities of the parent dyes could not be entirely accounted for by the potencies of their respective naphthylamine derivatives. Taken together, these findings extend previous observations of the superior metabolic activation capabilities of hamster, relative to rat hepatocytes, and further demonstrate the utility of testing chemicals in both the hamster and rat HPC/DR assays.

Relative susceptibility of microsomes from lung, heart, liver, kidney, brain and testes to lipid peroxidation: correlation with vitamin E content.

Rates of in vitro lipid peroxidation of microsomes and homogenates were found to vary widely among different tissues and species. In rats and rabbits, lung microsomes peroxidized at a 25- to 50-fold lower rate than liver, kidney, testes and brain microsomes. Heart microsomes peroxidized at a rate slightly greater than, but most similar to, lung microsomes. Comparison of tissue homogenates also revealed the unique resistance of lung and heart to lipid peroxidation. The ratio of vitamin E to peroxidizable polyunsaturated fatty acids in lung and heart microsomes was several-fold higher than in microsomes from the other tissues studied, which accounted for the relative resistance of lung and heart to lipid peroxidation. Liposomes of extracted rat lung microsomal lipid were also resistant to peroxidation and the amount of vitamin E contained in the lung lipid extract was sufficient to confer the same degree of resistance when incorporated into an equivalent amount of rat liver lipid. Higher rates of peroxidation in mouse lung microsomes relative to rabbit, rat and human lung microsomes were similarly correlated with a lower ratio of vitamin E to peroxidizable fatty acids in mouse lung microsomes. These data provide strong support for the role of vitamin E as the major cellular antioxidant, especially in the highly oxygenated tissues of heart and lung, and demonstrate the utility of the microsomal system in characterizing tissue differences in susceptibility to peroxidative membrane decomposition.

Comparison of rat and hamster hepatocyte primary culture/DNA repair assays.

Previous studies have demonstrated marked differences in the capacity of hepatocytes from rats or hamsters to mediate the metabolic activation of chemical carcinogens to genotoxic (ie, mutagenic) products. Thus far, very few investigations of species differences in DNA repair have been performed. Therefore, a comparison of the relative extent of DNA repair elicited by various genotoxic chemicals in rat and hamster hepatocytes was conducted, using the hepatocyte primary culture/DNA repair (HPC/DR) assay. Of the 11 chemicals tested, eight were more potent in inducing DNA repair in hamster hepatocytes than in rat hepatocytes. Dimethylnitrosamine, diethylnitrosamine, 2-acetylaminofluorene, 9-aminoacridine, pararosaniline hydrochloride, 1-naphthylamine, benzidine and 1,2:3,4-diepoxybutane were all active in hamster hepatocytes at a concentration at least ten times less than the lowest effective concentration in rat hepatocytes. The direct-acting alkylating agent, methylmethane sulfonate, was equipotent in inducing DNA repair in both rat and hamster hepatocytes, indicating that the differences in DNA repair observed for the other chemicals were probably not a result of species differences in DNA repair capacities. In contrast, 1-nitropyrene produced a greater DNA repair response in rat hepatocytes than hamster hepatocytes, while the bacterial mutagen 3-(chloromethyl)pyridine hydrochloride was inactive in both hepatocyte systems. These studies demonstrate the feasibility of using hamster hepatocytes in the HPC/DR assay and illustrate the utility of performing the assay with hepatocytes from more than one species.

Glutathione depletion by methyl chloride and association with lipid peroxidation in mice and rats.

Inhalation of methyl chloride (CH3Cl) by male B6C3F1 mice resulted in a concentration-dependent depletion of glutathione (GSH) in liver, kidney, and brain. Exposure for 6 hr to 100 ppm CH3Cl decreased the concentration of GSH in mouse liver by 45%, while exposure to 2500 ppm for 6 hr lowered liver GSH to approximately 2% of control levels. For those exposures which decreased liver GSH to less than 20% of control levels, the extent of liver GSH depletion was closely correlated with the capacity of a 9000g supernatant fraction from the liver to undergo lipid peroxidation in vitro. GSH was depleted to a lesser extent in mouse brain and kidney, compared to liver, and no relationship to peroxidation was observed for single exposures to CH3Cl. A dose-dependent decrease in liver GSH was also produced by diethyl maleate, although a nearly lethal amount (2 ml/kg) was required to lower liver GSH to less than 10% of control levels. Under these conditions the amount of lipid peroxidation was 3.5-fold less than in mice exposed to 2000 ppm CH3Cl. Exposure of rats to 2000 ppm CH3Cl reduced liver GSH to 20% of control levels, compared to 4.5% in mice similarly exposed, and under these exposure conditions the amount of lipid peroxidation measured in vitro was 40-fold greater in mouse liver than in rat liver. During exposure of mice to 2500 ppm CH3Cl, ethane expiration increased to an extent comparable to that produced by administration of 2 ml/kg of CCl4. These findings suggest that GSH depletion in liver may be an important component of CH3Cl-induced hepatotoxicity.

Effect of silica and volcanic ash on the content of lung alveolar and tissue phospholipids.

Silica or volcanic ash (VA) was administered to rats via intratracheal instillation and the changes in extracellular (i.e., lavage fluid) and tissue phospholipids, as well as various biochemical parameters, were monitored over a 6-month period. VA produced relatively minor (up to 2.8-fold) increases in lung tissue or lavage fluid phospholipids that were maximal at 1 month postinstillation. These increases were quantitatively similar to the increases in protein and DNA content of lung tissue and lavage fluid induced by VA and, thus, may be attributable to hypercellularity and accumulation of cellular breakdown products in the alveolar lumen. Instillation of silica produced a much greater (up to 11-fold) increase than VA in total phospholipid over time, primarily due to a 14-fold increase in phosphatidylcholine (PC). The accumulation of PC was more pronounced in the lavage fluid during the first month following silica instillation, but thereafter progressed more rapidly in the lung tissue. The relatively small increases (1.3- to 3.5-fold) in other phospholipids induced by silica appeared to be nonspecific, since they did not differ greatly from the increases in lung weight, DNA, and protein. Collectively, these results indicate that intratracheal instillation of silica induces selective accumulation of lung PC, implying enhanced synthesis and secretion of pulmonary surfactant from alveolar epithelial Type II cells into the lumen.

The role of glutathione and cytochrome P-450 in the metabolism of methyl chloride.

Rat liver microsomes metabolized methyl chloride to formaldehyde at a rate 15-fold less than the rate of benzamphetamine demethylation. The reaction rate was stimulated approximately 2-fold in microsomes from phenobarbital-pretreated rats and was inhibited by addition of SKF-525A, carbon monoxide, metyrapone, and hexobarbital to the microsomal suspension, indicating dependence on cytochrome P-450. The in vivo incorporation of 14CH3Cl into liver macromolecules, previously shown to reflect metabolism to CH3Cl to formate, was not significantly altered by SKF-525A, Aroclor 1254, or 3-methylcholanthrene pretreatment of rats, although pretreatment with phenobarbital produced a 35 and 28% increase in 14CH3Cl uptake into liver lipid and acid-insoluble material, respectively. Pretreatment with phenobarbital increased the in vivo metabolism of 14CH3Cl to 14CO2 (also derived from a formate intermediate) by 19%, but had no effect on urinary metabolites derived from 14CH3Cl. SKF-525A inhibited 14CO2 production from 14CH3Cl by 30% and also had no effect on urinary excretion of 14C. In contrast, pretreatment with diethylmaleate inhibited 14CH3Cl incorporation into liver macromolecules by 70 to 85%, and lowered 14CO2 expiration and urinary 14C excretion by 52 and 60%, respectively. S-Methylcysteine pretreatment produced a similar inhibition of 14CH3Cl incorporation and metabolism to 14CO2; urinary excretion of 14C, however, was approximately doubled. 14CH3Cl uptake into liver was also stimulated by cysteine pretreatment. These results indicate a strong dependence of CH3Cl metabolism on tissue nonprotein sulfhydryl content and suggest a possible role for cytochrome P-450 in the in vivo metabolism of CH3Cl. A scheme for the metabolism of CH3Cl is postulated which involves initial reaction with glutathione, and sequential metabolism of the conjugate to S-methylcysteine, methanethiol, and formaldehyde.

Variations on the standard protocol design of the hepatocyte DNA repair assay.

Several variations on the standard primary rat hepatocyte DNA/repair assay were evaluated for their ability to enhance the sensitivity of this genotoxicity test system. The use of hamster hepatocytes proved to be a much more sensitive system than rat hepatocytes for detecting the DNA repair inducing ability of the nitrosamines, dimethylnitrosamine and diethylnitrosamine, and the aromatic amines, 2-acetylaminofluorene, 9-aminoacridine, 1-naphthylamine and benzidine. In addition, hamster hepatocytes were a more sensitive indicator of the genotoxicity of the azo dyes, o-aminoazotoluene, Congo Red and Evans Blue. However, the azo reduction product of the azo dyes Congo Red, Trypan Blue and Evans Blue, benzidine and o-tolidine, respectively, were active in both rat and hamster hepatocytes at concentrations that were 10-100 fold lower than the parent dyes. This suggests that little or no azo reduction of the dyes occurred in the in vitro assay systems. The in vivo-in vitro variation of the rat hepatocytes DNA/repair assay exhibited a positive DNA repair response with the azo dye solvent Yellow 5, which was negative in the standard in vitro assay. The in vivo-in vitro hepatocyte DNA repair assay was also more sensitive for detecting the genotoxic activity of Evans Blue, which was positive in the in vivo-in vitro assay and equivocal in the standard in vitro assay. Also, Solvent Yellow 14 was negative in the in vitro assay, but induced an equivocal DNA repair response in the in vivo-in vitro assay system. A treatment/3H-thymidine labeling period of approximately 18 hours, compared to 4 hours, was demonstrated to be superior for detecting the DNA repair elicited by the mutagens 4-nitroquinoline-1-oxide, mitomycin C, dimethylnitrosamine and methyl methanesulfonate in the in vitro rat hepatocyte assay. There was little or no difference observed between the 4 hour and 18 hour treatment/labeling incubation periods for the detection of DNA repair induced by 2-acetylaminofluorene, aflatoxin B1, and benzidine. The data suggest that these several variations on the standard rat hepatocyte DNA/repair assay should be considered when evaluating the genotoxicity of chemicals for safety purposes.

Evaluation of the toxicity of ISIS 2302, a phosphorothioate oligonucleotide, in a 4-week study in CD-1 mice.

The subchronic toxicity of ISIS 2302 and ISIS 3082, phosphorothioate oligonucleotides with antisense activity against human and murine ICAM-1 mRNA, respectively, was investigated in CD-1 mice. ISIS 2302 is currently in clinical trials as an anti-inflammatory agent. Because of the differences in mRNA sequence targets between humans and mice, ISIS 2302 has no pharmacologic activity in mice. ISIS 3082 was specifically designed to inhibit murine ICAM-1 and was included in this study to evaluate the effects of prolonged ICAM-1 inhibition. The oligonucleotides were administered by bolus i.v. injection (via tail vein) every other day for 27 days (14 doses) at dose levels of 0, 0.8, 4, 20, and 100 mg/kg per injection ISIS 2302 or 20 mg/kg per injection ISIS 3082. The basic group size consisted of 10 male and 10 female mice, which were sacrificed 2 days after the last dose and an additional 5 mice per sex in vehicle control and 100 mg/kg ISIS 2302 dose groups, which remained on study for a 28-day treatment-free period. No treatment-related deaths occurred during this study, and there were no effects of either oligonucleotide on body weight gain or food consumption. The most common changes observed in this study included a mixed mononuclear cell infiltrate seen in a number of organs or tissues, splenomegaly, and lymphoid hyperplasia at dose levels of > or = 20 mg/kg ISIS 2302. In the group that received the highest dose level of ISIS 2302 (100 mg/kg), there were alterations in serum chemistry parameters that appeared to be related to perturbations in the liver, including 3- to 4-fold increases in aspartate and alanine aminotransferase and smaller changes in bilirubin, alkaline phosphatase, cholesterol, triglycerides, and albumin levels. Treatment-related effects on hematologic parameters were limited to the 100 mg/kg ISIS 2302 dose group and included slight monocytosis and thrombocytopenia. None of the effects observed appeared to be life threatening. Complete or partial reversal of all effects was evident in the remaining high-dose ISIS 2302 animals at the end of the 4-week recovery period. Comparison of the effects produced by the same dose level (20 mg/kg) of ISIS 2302 and ISIS 3082 did not reveal any differences that could be attributed to exaggerated pharmacology. In conclusion, treatment-related alterations were observed primarily at the 100 mg/kg dose level, including immune stimulation and hepatic alterations, which were partially reversed following a 4-week treatment-free period.

Inhibition of coagulation by a phosphorothioate oligonucleotide.

In the development of antisense therapeutics, there have been a number of hybridization-independent effects characterized for phosphorothioate oligodeoxynucleotides. One such effect is the transient prolongation of clotting times following intravenous infusion of high doses. In this study, inhibition of clotting times was characterized by determining the time course of both APTT and plasma oligonucleotide following intravenous infusion of ISIS 2302 in cynomolgus monkeys. Prolongation of APTT was also achieved by addition of ISIS 2302 to citrated blood from untreated monkeys, allowing the investigation of the mechanism of inhibition in vitro. Results from this study clearly indicate that the intrinsic pathway (APTT) was more sensitive to inhibition than the extrinsic pathway (PT). The prolongation of APTT was also shown to be transient and closely correlated with plasma oligonucleotide concentrations. The extent of APTT prolongation can be controlled by minimizing peak plasma oligonucleotide concentrations through lowering the dose or prolonging infusion duration. Direct addition of ISIS 2302 to blood produced quantitatively similar inhibition of clotting times. This effect was similar for a number of different phosphorothioate oligodeoxynucleotides, but oligonucleotides containing phosphodiester linkages and 2'-propoxy linkages were much less inhibitory. Additional in vitro studies indicated that the mechanism of inhibition was independent of that of heparin and possibly involved selective inhibition of the intrinsic pathway as well as the common clotting pathway. Investigation of selective clotting factors indicated that there was no direct inhibition of the enzymatic activity of factor Xa, XIa, or thrombin using chromogenic substrates. However, ISIS 2302 did produce a concentration-dependent increase in clotting time when fibrinogen was used as the substrate for thrombin.

Effect of di(2-ethylhexyl) phthalate on DNA repair and lipid peroxidation in rat hepatocytes and on metabolic cooperation in Chinese hamster V-79 cells.

Experiments were conducted to test the hypothesis that the hepatocarcinogenicity of di(2-ethylhexyl) phthalate (DEHP) is due to its ability to produce DNA damage, either directly or as a result of the proliferation of peroxisomes and accompanying increased production of H2O2 and other DNA--damaging oxygen radicals induced by sustained exposure to the plasticizer. DNA repair, as assessed by the autoradiographic measurement of unscheduled DNA synthesis (UDS), was not observed in primary rat hepatocytes exposed in vitro to 10(-5)-10(-2) M DEHP or in vivo by a single gavage dose of 5 g DEHP/kg body weight administered 2, 15, or 24 h prior to the isolation of hepatocytes. Thus, DEHP does not appear to directly produce repairable DNA damage in rat hepatocytes. Sustained feeding of DEHP at a dietary concentration of 2% led to a marked proliferation of peroxisomes in the liver after 4 wk. Additional administration of a single gavage dose of 5 g DEHP/kg body weight to animals fed the 2% diet for 4 or 8 wk, as well as to 4-wk-fed animals that were also pretreated with 3-amino-1,2,4-triazole to inhibit endogenous catalase activity, did not induce any detectable DNA repair in hepatocytes isolated 15 h following the single gavage dose of DEHP. Lipid peroxidation measured in the 9000 X g supernatant of livers from animals treated with a single dose of 5 g DEHP/kg body weight or the 2% DEHP diet for 6 wk plus a single dose of 5 g/kg body weight did not differ from controls. These findings suggest that DEHP does not elicit DNA damage or lipid peroxidation in liver consequent to the proliferation of peroxisomes resulting from prolonged administration. In addition, at noncytotoxic concentrations DEHP failed to produce a positive response in the Chinese hamster V-79 metabolic cooperation assay for tumor promoters.

Single-dose hemodynamic toxicity and pharmacokinetics of a partial phosphorothioate anti-HIV oligonucleotide (AR177) after intravenous infusion to cynomolgus monkeys.

5' GTGGTGGGTGGGTGGGT-3' (AR177) is a 17-mer oligonucleotide with anti-human immunodeficiency virus (HIV) activity that is composed of a phosphodiester backbone and single phosphorothioate linkages at the 3' and 5' ends. A hemodynamic toxicity study was conducted in which cynomolgus monkeys were infused i.v. over a 10-minute period with single doses of 5, 20 or 50 mg AR177/kg or saline. Blood pressure, ECG, clinical chemistry, hematology, complement factors, coagulation parameters and the AR177 plasma concentration were determined. AR177 did not cause any mortality in this study, nor did it cause changes in blood pressure, ECG, clinical chemistry or hematology parameters at any dose. There was a minimal, dose-dependent increase in the levels of complement split product Bb and total hemolytic complement. 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 after infusion. The time course of the inhibition of coagulation closely matched the plasma levels of AR177. There was a no-effect plasma AR177 concentration vs. activated partial thromboplastin time of approximately 60 to 100 micrograms AR177/ml, above which there was prolongation of activated partial thromboplastin time. These data demonstrate that AR177 does not cause significant hemodynamic toxicity at the doses studied and that this drug could be administered as a rapid infusion without any acute, life-threatening effects at doses that produce plasma concentrations that have shown anti-HIV activity in vitro.

Repeat-dose toxicity and pharmacokinetics of a partial phosphorothioate anti-HIV oligonucleotide (AR177) after bolus intravenous administration to cynomolgus monkeys.

5'GTGGTGGGTGGGTGGGT-3' (AR177) is a partial phosphorothioate, 17-mer oligonucleotide that has been shown to have anti-human immunodeficiency virus (HIV) activity in vitro and to be a potent inhibitor of HIV-1 integrase. A repeat-dose toxicity and pharmacokinetic study was conducted in which cynomolgus monkeys were given bolus i.v. injections of 2.5, 10 or 40 mg AR177/kg/day every other day for a total of 12 doses. Control monkeys received saline. ECG, clinical chemistry, hematology, coagulation parameters, histopathology and the AR177 plasma concentration were evaluated. AR177 did not cause any mortality in this study, nor did it cause changes in ECG, clinical chemistry, hematology values or histology. However, there was a dose-dependent inhibition of coagulation measured by a prolongation of activated partial thromboplastin time; this inhibition was reversible with drug washout. Analysis of plasma samples by HPLC demonstrated that there was no difference between the AR177 plasma concentrations that were achieved after the 1st and 12th (last) doses of 2.5, 10 or 40 mg/kg. There was a direct relationship between the AR177 plasma concentration and activated partial thromboplastin time. These results indicate that repeated bolus i.v. administration of AR177 to cynomolgus monkeys at doses as high as 40 mg/kg was well tolerated and was not associated with the serious cardiovascular responses previously observed with other oligonucleotides administered i.v.

Activation of the alternative pathway of complement by a phosphorothioate oligonucleotide: potential mechanism of action.

Intravenous infusion of high doses of phosphorothioate oligonucleotides in monkeys has been associated with transient alterations in hematologic and hemodynamic parameters, which appear to be secondary to complement activation. ISIS 2302, a phosphorothioate oligonucleotide specific for human intracellular adhesion molecule-1, was used to further characterize complement activation in monkeys. Complement activation occurred selectively through the alternative pathway resulting in increased plasma concentrations of the complement split products Bb, C3a and C5a. Marked fluctuations in circulating neutrophil counts and reductions in cardiac output were closely associated with peak production of anaphylatoxins C3a and C5a. Changing both dose and infusion duration revealed that complement activation is related to plasma levels of oligonucleotide, and that there is a minimum threshold concentration of approximately 50 micrograms/ml of ISIS 2302 that is required to activate complement. Dose regimens in which plasma concentrations do not exceed this threshold do not result in complement activation. Further investigation reveals that plasma concentrations of a key regulatory component of the alternative pathway, Factor H, were also decreased after administration of ISIS 2302. Decreases in Factor H levels are suggestive of a possible mechanism of complement activation. Direct interaction between ISIS 2302 and Factor H was demonstrated in a competition assay, where increasing concentrations of ISIS 2302 eluted Factor H from a heparin-sepharose column. These data demonstrate a clear correlation between plasma oligonucleotide concentrations and complement activation. Interactions between ISIS 2302 and Factor H may lead to activation of the alternative complement pathway.

Toxicity and toxicokinetics of a phosphorothioate oligonucleotide against the c-myc oncogene in cynomolgus monkeys.

A 2-week toxicity and toxicokinetic study of a 15-mer phosphorothioate oligonucleotide, INX-3280, against the c-myc oncogene was performed in cynomolgus monkeys. As this oligonucleotide readily adopts an aggregate structure, a quadruplex, which may be associated with adverse physiologic effects, this study was performed using INX-3280 that had been converted to its monomeric form. Animals received intravenous (i.v.) infusions of monomeric INX-3280 three times per week for 2 weeks at doses of 3 or 15 mg/kg per administration. The monkeys were examined for clinical signs: changes in hematology, serum chemistry, coagulation, and urinalysis parameters; complement activation; macroscopic findings at necropsy; and histopathologic alterations. In addition, the toxicokinetics of INX-3280 were evaluated, using a validated HPLC assay, after the first and last (sixth) doses. No treatment-related clinical signs of any adverse effects were observed, and there were no test article-related changes in hematology, serum chemistry, or complement activation parameters. The only alteration in clinical pathology parameters was a minor (30%) prolongation of the activated partial thromboplastin time (aPTT), reflecting slight inhibition of the intrinsic coagulation pathway, which was less than that reported with other oligonucleotides given at similar doses. Treatment-related histopathologic alterations consisted of characteristic accumulation of basophilic material in the cytoplasm of tubular epithelial cells in the kidney, resident macrophages in the lymph nodes, and Kupffer cells in the liver. These changes were graded as minimal in all cases. The basophilic material is believed to reflect accumulation of the oligonucleotide or metabolites or both. The pharmacokinetic parameters of INX-3280 were identical on the first and sixth administrations and were similar to those reported for other phosphorothioate oligonucleotides. Maximum concentration (Cmax) values for INX-3280 (101-119 microg/ml) were in excess of the threshold plasma concentrations reported to trigger complement activation by phosphorothioate oligonucleotides. It is concluded that the safety profile of monomeric INX-3280 in cynomolgus monkeys is quite favorable relative to the known effects of other phosphorothioate oligonucleotides, particularly with respect to the blood level-related toxicities of this class of compounds, including complement activation and inhibition of coagulation. This study found no toxicities that were expected to be clinically significant.

Subchronic toxicity studies with the leukotriene D4 antagonist RG 12525.

Preclinical safety studies with the leukotriene D4 antagonist RG 12525 were conducted by the oral route in mice, rats, and monkeys. Oral administration of RG 12525 was repeated daily in studies up to 6 months in duration. RG 12525 was shown to have limited high-dose toxicity after repeated oral administration. The effects of RG 12525 were strongly dependent upon the species considered. High doses of RG 12525 caused significant increases in liver weight in mice, rats, and monkeys that were associated with diffuse hepatocellular hypertrophy in mice and rats but not in monkeys. No related clinical chemistry changes were observed in any of the species and hepatic activities of peroxisomal enzymes or cytochrome P450 were increased only slightly. Proliferation of brown adipose tissue (BAT) was observed in rats and mice but not in monkeys. The BAT reaction was more pronounced in the interscapular area but it was also observed in other subcutaneous locations as well as in mediastinal and bone marrow fat. In all locations, the RG 12525-induced BAT had some morphological similarities with cold-adapted BAT. Repeated administration of RG 12525 at high doses to female rats resulted in a lack of progression to the luteal phase of the estrous cycle that was reversible after discontinuation of treatment. Finally, RG 12525 was nephrotoxic in mice with males being more sensitive than females.

Toxicity of the HMG-coenzyme A reductase inhibitor, lovastatin, to rabbits.

Lovastatin, a specific inhibitor of the rate-limiting enzyme in cholesterol biosynthesis, HMG-CoA reductase, has been shown to be highly effective in lowering serum cholesterol in animals and humans and thus represents a promising approach to the treatment and prevention of cardiovascular disease. During the preclinical safety assessment of lovastatin, oral doses that were tolerated by dogs, rats and mice were found to be lethal to rabbits in subacute studies. Postmortem findings in rabbits consisted of centrilobular hepatic necrosis, frequently accompanied by renal tubular necrosis and occasionally gallbladder necrosis. The liver lesions were associated with up to 300-fold elevations in serum aspartate and alanine aminotransferase activities, whereas the kidney lesions resulted in accumulations of serum urea nitrogen and creatinine. The organ damage was preceded by a progressive decline in food consumption and loss of body weight. All histopathological and serum biochemical changes induced by lovastatin were completely prevented by coadministration of mevalonate, the product of the inhibited HMG-CoA reductase enzyme. In addition, administration of mevalonate after the onset of lovastatin-induced hepatotoxicity effectively reversed the toxicity despite continued drug treatment. These findings indicated that the toxicity of high doses of lovastatin to rabbits is a consequence of a highly exaggerated pharmacologic action in blocking mevalonate synthesis. However, supplementation of lovastatin-treated rabbits with oral doses of the major product of mevalonate metabolism, cholesterol, paradoxically enhanced the liver and kidney damage, which suggested that the toxicity of lovastatin stemmed from depletion of a nonsterol metabolite(s) of mevalonate critical for cell viability.