Intraesophageal administration of GS-nitroxide (JP4-039) protects against ionizing irradiation-induced esophagitis

Abstract

Background/aim: this study evaluated esophageal radioprotection by the Gramicidin S (GS) derived-nitroxide, JP4-039, a mitochondrial targeting peptide-isostere covalently-linked to 4-amino-Tempo, delivered in a novel swallowed oil-based (F15) formulation.

Materials and methods: C57BL/6HNsd female mice received intraesophageal F15 formulation containing JP4-039 (4 mg/ml in 100 microl volumes) 10 minutes before 28 or 29 Gy upper body irradiation compared to MnSOD-PL (100 microl containing 100 microg plasmid) 24 hours prior to irradiation. Subgroups received 1 × 10(7) C57BL/6HNsd, GFP(+) male bone marrow cells intravenously 5 days after irradiation.

Results: JP4-039/F15 or MnSOD-PL increased survival compared to irradiated controls (p<0.0001 for either). Marrow injection further increased survival (p=0.0462 and 0.0351, respectively). Esophagi removed at 1, 3, 7, 14, 24, or 60 days showed bone marrow-derived cells in the esophagi.

Conclusion: intraesophageal GS-nitroxide radioprotection is mediated primarily through recovery of endogenous esophageal progenitor cells.

Figure 1

Pharmacokinetics of clearance of JP4-039 intravenously injected into C57BL/6JHNsd mice. Mice were injected with 4 mg/kg JP4-039 in cremphor EL/ethanol 50% to 50%. Serum samples were collected and assayed. Each symbol represents an individual mouse. The methods for assay of nitroxide by EPR have been published previously (38, 39).

Figure 2

Superior penetration of cationic multilamellur liposome F15 containing 0.5 mole percent of Lissamine Rhodamine B-DOPE into the murine esophagus by swallowed F15 as compared to control formulation that did not contain dioleylamine amido-L-glutamate. Images of esophageal cross-sections taken at 10 minutes after swallow of 4 mg/kg of protein in 100 μl formulation are shown (magnification: ×100). A: F15 formulation; B: Control formulation.

Figure 3

Quantitation of mitochondrial targeted nitroxide JP4-039 for several time points over a 60-minute period after swallow in the esophagus by EPR. The results represent mean and standard error of n=5 per group. Controls included non phycoerythrin-treated esophagi. The experimental procedures are described in Materials and Methods and in (38, 39).

Figure 4

Effect of JP4-039/F15 on esophageal irradiation toxicity. Female mice (15 per group) received MnSOD-PL, JP4-039 in F15 formulation, F15 formulation, or 29 Gy upper body irradiation alone as described in the Materials and Methods. P-Values showed a significant effect of pre-irradiation intraesophageal MnSOD-PL or JP4-039/F15 compared to F15 emulsion alone against 29 Gy.

Figure 5

Effect of GFP⁺ male marrow intravenous injection and JP4-039/F15 on esophageal irradiation toxicity. Female mice (15 per group) received 29 Gy upper body irradiation on day 0, then on day 5 they received1 ×10⁷ GFP⁺ marrow cells intravenously from male donors. P-values showed a significant effect of pre-irradiation intraesophageal MnSOD-PL or JP4-039/F15 on increasing the survival; p=0.0315 and p=0.0462, respectively.

Figure 6

Detection of GFP⁺ marrow-derived cells in the irradiated mouse esophagus after intravenous transplant. Mice were irradiated to 29 Gy to the esophagus on day 0, and then injected with 1×10⁷ GFP⁺ marrow cells intravenously on day 5 according to published methods (5-7). Five esophagus samples were removed from each animal in the various subgroups on days 1 (A), 3 (B), 7 (C), 14 (D), 28 (E) and 60 (F) after marrow injection. Samples of excised esophagi were prepared as single cell suspensions and then analyzed by cell sorting for GFP⁺ cells/10⁶ esophagus cells. Each symbol represents one esophagus.