Lack of toxicity of a STAT3 decoy oligonucleotide - PubMed (original) (raw)

doi: 10.1007/s00280-008-0823-6. Epub 2008 Sep 3.

Patricia J Tosca, Christa Zwayer, Michael J Ryan, Jerry D Johnson, Katherine A B Knostman, Patricia C Giclas, James O Peggins, Joseph E Tomaszewski, Timothy P McMurray, Changyou Li, Michael S Leibowitz, Robert L Ferris, William E Gooding, Sufi M Thomas, Daniel E Johnson, Jennifer R Grandis

Affiliations

Lack of toxicity of a STAT3 decoy oligonucleotide

Malabika Sen et al. Cancer Chemother Pharmacol. 2009 May.

Abstract

Background: STAT3 overexpression has been detected in several cancers including head and neck squamous cell carcinoma (HNSCC). Previous studies using intratumoral administration of a STAT3 decoy oligonucleotide that abrogates STAT3-mediated gene transcription in preclinical cancer models have demonstrated antitumor efficacy. This study was conducted to observe the toxicity and biologic effects of the STAT3 decoy in a non-human primate model, in anticipation of initiating a clinical trial in HNSCC patients.

Methods: Three study groups (two monkeys/sex/group) were administered a single intramuscular injection of low dose of STAT3 decoy (0.8 mg total dose/monkey), high dose of STAT3 decoy (3.2 mg total dose/monkey) or vehicle control (PBS alone) on day 1 and necropsies were performed on days 2 and 15 (one monkey/sex/group/day). Low and high doses of the decoy were administered in the muscle in a volume of 0.9 ml. Tissue and blood were harvested for toxicology and biologic analyses.

Results: Upon observation, the STAT3 decoy-treated animals exhibited behavior that was similar to the vehicle control group. Individual animal body weights remained within 1% of pretreatment weights throughout the study. Hematological parameters were not significantly different between the control and the treatment groups. Clinical chemistry fluctuations were considered within normal limits and were not attributed to the STAT3 decoy. Assessment of complement activation breakdown product (Bb) levels demonstrated no activation of the alternative pathway of complement in any animal at any dose level. At necropsy, there were no gross or microscopic findings attributed to STAT3 decoy in any organ examined. STAT3 target gene expression at the injection site revealed decreased Bcl-X(L) and cyclin D1 expression levels in the animals treated with high dose of STAT3 decoy compared to the animals injected with low dose of STAT3 decoy or the vehicle as control.

Conclusion: Based on these findings, the no-observable-adverse-effect-level (NOAEL) was greater than 3.2 mg/kg when administered as a single dose to male and female Cynomolgus monkeys. Plans are underway to test the safety and biologic effects of intratumoral administration of the STAT3 decoy in HNSCC patients.

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Figures

Fig. 1

Fig. 1

Apparent lack of effect of STAT3 decoy on body weight. Body weight measurements of the monkeys injected with PBS as the vehicle control (a), low dose (0.8 mg) of STAT3 decoy (b) and high dose (3.2 mg) of STAT3 decoy (c) were determined during the pretreatment period, on day 1 before treatment and on days 2 8 and 15 after STAT3 decoy treatment. No statistically significant difference in body weight was observed between the treatment groups at each time point. Each line represents an individual animal

Fig. 2

Fig. 2

STAT3 decoy administration may not affect serum chemistry or hematological parameters in monkeys. a Distribution of serum chemistries and b hematological indices in the blood of monkeys injected with the STAT3 decoy. Four monkeys were injected with PBS as control. Four monkeys were injected with the low dose of STAT3 decoy (0.8 mg) and four monkeys were injected with the high dose of STAT3 decoy (3.2 mg) and blood was collected from each animal at the times indicated. Data for each constituent are shown in pairs (top to bottom). The top graph is the day 2 profile for each monkey, as a line connecting each pretreatment to day 2. The lower graph is the magnitude of the change to day 2 by group. Line codes are solid line for control, small dashes for 0.8 mg STAT3 decoy and big dashes for 3.2 mg STAT3 decoy. The lower graph of each pair may include a dashed line to depict zero change

Fig. 2

Fig. 2

STAT3 decoy administration may not affect serum chemistry or hematological parameters in monkeys. a Distribution of serum chemistries and b hematological indices in the blood of monkeys injected with the STAT3 decoy. Four monkeys were injected with PBS as control. Four monkeys were injected with the low dose of STAT3 decoy (0.8 mg) and four monkeys were injected with the high dose of STAT3 decoy (3.2 mg) and blood was collected from each animal at the times indicated. Data for each constituent are shown in pairs (top to bottom). The top graph is the day 2 profile for each monkey, as a line connecting each pretreatment to day 2. The lower graph is the magnitude of the change to day 2 by group. Line codes are solid line for control, small dashes for 0.8 mg STAT3 decoy and big dashes for 3.2 mg STAT3 decoy. The lower graph of each pair may include a dashed line to depict zero change

Fig. 2

Fig. 2

STAT3 decoy administration may not affect serum chemistry or hematological parameters in monkeys. a Distribution of serum chemistries and b hematological indices in the blood of monkeys injected with the STAT3 decoy. Four monkeys were injected with PBS as control. Four monkeys were injected with the low dose of STAT3 decoy (0.8 mg) and four monkeys were injected with the high dose of STAT3 decoy (3.2 mg) and blood was collected from each animal at the times indicated. Data for each constituent are shown in pairs (top to bottom). The top graph is the day 2 profile for each monkey, as a line connecting each pretreatment to day 2. The lower graph is the magnitude of the change to day 2 by group. Line codes are solid line for control, small dashes for 0.8 mg STAT3 decoy and big dashes for 3.2 mg STAT3 decoy. The lower graph of each pair may include a dashed line to depict zero change

Fig. 2

Fig. 2

STAT3 decoy administration may not affect serum chemistry or hematological parameters in monkeys. a Distribution of serum chemistries and b hematological indices in the blood of monkeys injected with the STAT3 decoy. Four monkeys were injected with PBS as control. Four monkeys were injected with the low dose of STAT3 decoy (0.8 mg) and four monkeys were injected with the high dose of STAT3 decoy (3.2 mg) and blood was collected from each animal at the times indicated. Data for each constituent are shown in pairs (top to bottom). The top graph is the day 2 profile for each monkey, as a line connecting each pretreatment to day 2. The lower graph is the magnitude of the change to day 2 by group. Line codes are solid line for control, small dashes for 0.8 mg STAT3 decoy and big dashes for 3.2 mg STAT3 decoy. The lower graph of each pair may include a dashed line to depict zero change

Fig. 3

Fig. 3

High-dose STAT3 decoy may reduce pSTAT3 levels in PBMCs of monkeys. PBMCs from the monkeys injected with vehicle control, low (0.8 mg) or high dose (3.2 mg) of STAT3 decoy were fixed, washed and stained with phosphoSTAT1 (pSTAT1) and phosphoSTAT3 (pSTAT3) antibodies. Single color flow cytometry was performed on a Beckman coulter flow cytometer. The upper panels demonstrate the mean fluorescence intensity (MFI) from pSTAT1 (left panel) and pSTAT3 (right panel) from PBMCs of each animal administered the STAT3 decoy. The lower panels demonstrate the change in pSTAT1 (left panel) and pSTAT3 (right panel) MFI in PBMC from animals administered with vehicle control, low dose 0.8 mg STAT3 decoy or high dose 3.2 mg STAT3 decoy

Fig. 4

Fig. 4

STAT3 decoy administration does not induce complement activation. Complement activation (breakdown product Bb) in the blood of monkeys injected with vehicle control, low (0.8 mg) or high dose (3.2 mg) of STAT3 decoy determined prior to treatment and after 24 h. Group medians are connected with a solid line. No significant difference in complement activation was observed between the control group and the low and high dose of STAT3 decoy injected groups

Fig. 5

Fig. 5

STAT3 decoy modulates target gene expressed at the injection site. a Whole cell lysates were prepared from the muscle tissue surrounding injection sites in control animals and animals treated with 0.8 mg or 3.2 mg STAT3 decoy. Proteins (40 µg /lane) were resolved on a 12.5% SDS/PAGE gel and subjected to immunoblotting with anti-Bcl-XL antibody. The blot was stripped and reprobed with anti-β-tubulin to assess protein loading. The experiment was performed twice, with similar results each time. b RNA was extracted from the muscle tissue of monkey injected with low (0.8 mg) and high dose (3.2 mg) of STAT3 decoy and PBS as the vehicle control and RT-PCR was performed to determine cyclin D1 level. Expression of cyclin D1 is relative to GAPDH level. The bar graph represents cumulative results from four samples per treatment group

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