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Identification of granulocyte colony-stimulating factor and interleukin-6 as candidate biomarkers of CBLB502 efficacy as a medical radiation countermeasure - PubMed

. 2012 Nov;343(2):497-508.

doi: 10.1124/jpet.112.196071. Epub 2012 Jul 26.

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Identification of granulocyte colony-stimulating factor and interleukin-6 as candidate biomarkers of CBLB502 efficacy as a medical radiation countermeasure

Vadim I Krivokrysenko et al. J Pharmacol Exp Ther. 2012 Nov.

Abstract

Given an ever-increasing risk of nuclear and radiological emergencies, there is a critical need for development of medical radiation countermeasures (MRCs) that are safe, easily administered, and effective in preventing and/or mitigating the potentially lethal tissue damage caused by acute high-dose radiation exposure. Because the efficacy of MRCs for this indication cannot be ethically tested in humans, development of such drugs is guided by the Food and Drug Administration's Animal Efficacy Rule. According to this rule, human efficacious doses can be projected from experimentally established animal efficacious doses based on the equivalence of the drug's effects on efficacy biomarkers in the respective species. Therefore, identification of efficacy biomarkers is critically important for drug development under the Animal Efficacy Rule. CBLB502 is a truncated derivative of the Salmonella flagellin protein that acts by triggering Toll-like receptor 5 (TLR5) signaling and is currently under development as a MRC. Here, we report identification of two cytokines, granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6), as candidate biomarkers of CBLB502's radioprotective/mitigative efficacy. Induction of both G-CSF and IL-6 by CBLB502 1) is strictly TLR5-dependent, 2) occurs in a CBLB502 dose-dependent manner within its efficacious dose range in both nonirradiated and irradiated mammals, including nonhuman primates, and 3) is critically important for the ability of CBLB502 to rescue irradiated animals from death. After evaluation of CBLB502 effects on G-CSF and IL-6 levels in humans, these biomarkers will be useful for accurate prediction of human efficacious CBLB502 doses, a key step in the development of this prospective radiation countermeasure.

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Figures

Fig. 1.
Fig. 1.

Dose-dependent effect of CBLB502 on 30-day survival of lethally irradiated mice. a, Kaplan-Meier curves illustrating mouse survival for 30 days after TBI. Female ICR mice (12/group) were injected intramuscularly with vehicle or one of the indicated doses of CBLB502 and irradiated with 10 Gy of TBI 30 min later. b, CBLB502 dose response of 30-day survival in the same study as shown in a, with filled markers indicating statistically significant difference versus vehicle control group (p < 0.05 by two-tailed Fisher's exact test). c, Kaplan-Meier curves illustrating mouse survival for 30 days after TBI. Forty female C57BL/6J mice per group were irradiated with 9.5 Gy of TBI and injected subcutaneously with either vehicle or the indicated doses of CBLB502 24 h later. d, CBLB502 dose response of 30-day survival in the same study shown in c, with filled markers indicating statistically significant difference versus vehicle control group (p < 0.05 by two-tailed Fisher's exact test).

Fig. 2.
Fig. 2.

G-CSF and IL-6 responses in nonirradiated mice. a and b, time-dependent changes in G-CSF (a) and IL-6 (b) plasma levels in nonirradiated female ICR mice injected intramuscularly with either vehicle or the indicated doses of CBLB502. Each data point represents the mean ± S.D. cytokine concentration measured in plasma of six mice. c and d, time-dependent changes in G-CSF (c) and IL-6 (d) plasma levels in nonirradiated female C57BL/6J mice injected subcutaneously with either vehicle or the indicated doses of CBLB502. Each data point represents the mean ± S.D. cytokine concentration measured in plasma of six mice. e and f, congruence of the CBLB502 dose response of survival and cytokine induction in ICR (e) and C57BL/6J (f) mice. For ICR mouse studies, the cumulative data from several similar experiments are presented. Cytokine data points represent natural logarithm of mean AUC(0–24) increases per dose group. Dashed line represents four-parameter sigmoid (Emax) curve fit for the survival data (represented as natural logarithm of survival odds ratio of treatment versus control group).

Fig. 3.
Fig. 3.

G-CSF and IL-6 responses in rhesus macaques and dogs. a, time-dependent changes in IL-6 plasma levels in nonirradiated male and female beagle dogs (n = 6; 1:1 sex ratio) injected intramuscularly with either vehicle or the indicated doses of CBLB502. Each data point represents the mean ± S.D. b and c, time-dependent changes in G-CSF (b) and IL-6 (c) plasma levels in nonirradiated male and female rhesus macaques (n = 6; 1:1 sex ratio) injected intramuscularly with either vehicle or the indicated doses of CBLB502. Each data point represents the mean ± S.D. d and e, CBLB502 dose-dependent changes in exposures (AUC(0–24)) to G-CSF (d) and IL-6 (e) in the three treated species: mouse, dog, and rhesus macaque. Mean data per dose group are shown. Dashed lines represent linear regression trend lines (in log-log coordinates).

Fig. 4.
Fig. 4.

Comparison of CBLB502 dose-dependent G-CSF and IL-6 exposures in nonirradiated versus irradiated mice and rhesus macaques. a and b, mean G-CSF (a) and IL-6 (b) exposures (AUC(0–24)) at indicated CBLB502 dose levels in ICR mice. c and d, mean G-CSF (c) and IL-6 (d) exposures (AUC(0–24)) at the indicated CBLB502 dose levels in rhesus macaques. Dashed lines represent linear regression trend lines (in log-log coordinates).

Fig. 5.
Fig. 5.

Influence of G-CSF or IL-6 neutralization on the radiomitigative efficacy of CBLB502 in mice. a, experimental scheme. Groups of 40 C57BL/6J female mice were exposed to 9 Gy of TBI. At +22 h relative to irradiation, the mice were injected intraperitoneally with anti-G-CSF, anti-IL-6, or control IgG antibodies and then injected intravenously with 60 μg/kg CBLB502 2 h later. The vehicle control group received 9 Gy of TBI and intravenous injection of the vehicle at +24 h. b, Kaplan-Meier curves illustrating mouse mortality kinetics for 30 days after TBI.

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