Subchronic Toxicities

6.8.3.2.1 Immune Stimulation. In the rodent, the most prominent toxicity is immune stimulation. This effect is also frequently a confounding variable that must be evaluated with regard to the mechanism of action of pharmacological effects (for review, see Ref. 265). Subchronic administration of doses as low as 10 mg kg-' day-1 in rodents results in splenomegaly, lymphoid hyperplasia, and diffuse multiorgan mixed mononuclear cell infiltrates (276-279). These effects are reminiscent of stimulator effects observed on isolated splenocytes (261, 280, 281). In fact, these in vitro studies are reasonably predictive of the relative potencies of phosphorothioates in moving in vivo immune stimulation. Immune stimulation in the rodent is a property common to all phosphorothioate oligodeoxynucle-otides, but potency varies substantially as a function of sequence. Rodent immune stimu-motifs include palindromic sequences and CG motifs (282,283).

In primates, immune stimulation is far less prominent than that in the rodent. In the monkey, doses necessary to produce immune stimulation have not been identified, despite evaluation of numerous oligonucleotides at a variety of doses and schedules (169). At least one factor contributing to these species differences is that the optimal rodent immune stimulator sequence is simple and immune stimulation is induced by all sequences. In contrast, in the primate, the optimal sequences are different and more complex (284) and the response is much less promiscuous with regard to sequence. Modifications of the base, the sugar, and the backbone can reduce immune stimulation in the rodent. For example, 5'-methycytosine and 2'-0-methoxyethyl containing oligonucleotides display substantially reduced potency for immune stimulation in the rodent (169).

6.8.3.2.2 Other Toxicities. Occasionally, in the rodent but not the monkey, single-cell hepatolyte neurons is observed and this has been related to immunostimulation (265). Also, on occasion, in the monkey transient thrombocytopenia is observed, perhaps associated with complement activation (for review, see Ref. 169). Other toxicities noted in animals are mild and occur infrequently at therapeutic doses. For example, occasional increases in liver function enyzmes are noted, but these occur at high doses and are not associated with histopathological changes (169).

6.8.4 Human Safety. At Isis, we have had the opportunity to study approximately 10 phosphorothioate oligonucleotidesin humans. We have studied antisense drugs administered intravitreally, intradermally, subcutaneously, intravenously, and topically. Vitravene, an in-travitreally administered drug, is commercially available around the world.

After systemic administration, in clinical trials, we have studied more than 2000 patients treated with more than 70.000 doses. Intravenous doses have ranged from 0.5 mg/kg every other day to as much as 30 mg kg-' day-1 as a continuous infusion. Most patients have been treated with multiple doses and many have been treated for prolonged periods (i.e., multiple months).

6,8.4.1 Complement Activation. With 2-h infusions at a dose of 2 mg/kg, no increases in complement-split products were observed in more than 300 patients with inflammatory diseases treated with ISIS 2302, a 20-nucleo-tide phosphorothioate antisense inhibitor of ICAM-1. Similarly, ISIS 5132, an inhibitor of C-raf kinase was dosed from 0.5 to 6.0 mg/kg and no meaningful increases in complement-split products were observed. ISIS 3521, a inhibitor gave equivalent data and both of these drugs were studied in patients with various malignant diseases. Similar data were observed with all the phosphorothioate oligodeoxynucleotides administered on this schedule in a variety of patients (for review, see Refs. 285 and 286). Three phosphorothio-ate oligodeoxynucleotides, ISIS 3521, a PKCa inhibitor; ISIS 5132, a C-raf kinase inhibitor; and ISIS 2503, a Ha-ras inhibitor, have been thoroughly characterized in patients with malignant diseases and dosed with long-term infusions. When given as 21 -day antitumor infusions at doses as high as 10 mgkg-' day \ no increases in complement-split products were observed (257, 287, 288). In contrast, when these drugs were given as 24-h infusions, significant increases in complement-split products were observed at doses of 18 mg kg-1 day-1 and greater (285). Despite these increases in complement-split products, only a small number of patients experienced mild fevers and myalgias at these very high doses.

6.8.4.2 Cytokines. At very high doses (i.e., 24 mg kg-' day-1) as a continuous i.v. infusion, significant increases in IL-6, IL-IR,, and were observed, once these often correlated with flulike syndromes (285). At present, the precise roles of each of the cytokines and complement activation in the clinical signs and symptoms (myalgia and fever) at high doses are not defined.

6.8.4.3 Coagulation. All phosphorothioate oligodeoxynucleotides studied in normal volunteers and in patients have resulted in transient, self-limited increases in activated partial thromboplastin time (aPTT). The effects are dose and peak plasma concentration dependent (285).These effects appear to be more prominent after the first dose. In none of the patients treated did we observe any evidence of bleeding, so the effects on aPTT have not proved to be a problem in the clinic and humans appear to behave similarly to other animals with regard to this side effect.

6.8.4.4 Platelet Effects. When phosphorothioate oligodeoxynucleotides have been administered by continuous i.v. infusion in patients with malignant diseases, transient thrombocytoplastin has been observed in a few patients (285). These effects were more frequent during the first course of therapy, were not obviously dose-related, and were not associated with bone marrow effects (253, 289). Most of the time, platelet counts returned to normal while dosing was continued and no bleeding was observed. The mecha nisms for these effects are not clear, but probably involve margination.

6.8.4.5 Other Toxicities. At no dose studied have significant effects on liver, kidney, or other organ performance been observed (285).

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