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. 2022 Oct 29;23(21):13191.
doi: 10.3390/ijms232113191.

Opaganib Protects against Radiation Toxicity: Implications for Homeland Security and Antitumor Radiotherapy

Lynn W Maines  1 , Randy S Schrecengost  1 , Yan Zhuang  1 , Staci N Keller  1 , Ryan A Smith  1 , Cecelia L Green  1 , Charles D Smith  1
Affiliations

Opaganib Protects against Radiation Toxicity: Implications for Homeland Security and Antitumor Radiotherapy

Lynn W Maines et al. Int J Mol Sci. .

Abstract

Exposure to ionizing radiation (IR) is a lingering threat from accidental or terroristic nuclear events, but is also widely used in cancer therapy. In both cases, host inflammatory responses to IR damage normal tissue causing morbidity and possibly mortality to the victim/patient. Opaganib, a first-in-class inhibitor of sphingolipid metabolism, has broad anti-inflammatory and anticancer activity. Opaganib elevates ceramide and reduces sphingosine 1-phosphate (S1P) in cells, conditions that increase the antitumor efficacy of radiation while concomitantly suppressing inflammatory damage to normal tissue. Therefore, opaganib may suppress toxicity from unintended IR exposure and improve patient response to chemoradiation. To test these hypotheses, we first examined the effects of opaganib on the toxicity and antitumor activity of radiation in mice exposed to total body irradiation (TBI) or IR with partial bone marrow shielding. Oral treatment with opaganib 2 h before TBI shifted the LD75 from 9.5 Gy to 11.5 Gy, and provided substantial protection against gastrointestinal damage associated with suppression of radiation-induced elevations of S1P and TNFα in the small intestines. In the partially shielded model, opaganib provided dose-dependent survival advantages when administered 4 h before or 24 h after radiation exposure, and was particularly effective when given both prior to and following radiation. Relevant to cancer radiotherapy, opaganib decreased the sensitivity of IEC6 (non-transformed mouse intestinal epithelial) cells to radiation, while sensitizing PAN02 cells to in vitro radiation. Next, the in vivo effects of opaganib in combination with radiation were examined in a syngeneic tumor model consisting of C57BL/6 mice bearing xenografts of PAN02 pancreatic cancer cells and a cross-species xenograft model consisting of nude mice bearing xenografts of human FaDu cells. Mice were treated with opaganib and/or IR (plus cisplatin in the case of FaDu tumors). In both tumor models, the optimal suppression of tumor growth was attained by the combination of opaganib with IR (± cisplatin). Overall, opaganib substantially protects normal tissue from radiation damage that may occur through unintended exposure or cancer radiotherapy.

Keywords: ABC294640; opaganib; radiation; sphingolipid; sphingosine kinase; xenograft.

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Conflict of interest statement

Lynn W. Maines, Randy S. Schrecengost, Yan Zhuang, Staci N. Keller, Ryan A. Smith, Cecelia L. Green and Charles D. Smith were employees at the time of the research and own stock in Apogee Biotechnology Corporation. Apogee Biotechnology Corporation owns patent rights to opaganib, and the value of these rights may be affected by the research reported in the enclosed paper. The funders (BARDA and the Department of Defense) assisted and approved the design of the experiments shown in Figure 4, Figure 5, Figure 6 and Figure 7, but had no role in the collection, analyses or interpretation of the data, in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Opaganib protects against lethality from total body irradiation. Mice in the vehicle groups were exposed to 7.5, 8.0, 8.5, 9.0 or 9.5 Gy TBI (Top Panel), and mice in the opaganib-treated groups (100 mg/kg oral, 1 h prior to radiation) were exposed to 9.5, 10.0, 10.5, 11.0 or 11.5 Gy TBI (Bottom Panel). Survival was monitored for 30 days.
Figure 2
Figure 2
Biodistribution and pharmacodynamics of opaganib in the small intestine. Top Panel: Mice were treated with opaganib (100 mg/kg) orally and sacrificed at either 3 or 7 h. Plasma and extracts from the small intestines were analyzed for opaganib concentrations as described in the Materials and Methods section. Middle and Bottom Panels: Mice were treated with 0 (○) or 100 (∎) mg/kg opaganib orally 2 h before exposure to 9.5 Gy radiation and then sacrificed at the indicated times for evaluation of TNFα and S1P levels in the small intestine (n = 3–5 mice per time point; * p < 0.05, ** p < 0.01, *** p < 0.001).
Figure 3
Figure 3
Effects of opaganib and radiation on the morphology of the small intestine. C57BL/6 mice were treated with 0 or 100 mg/kg opaganib orally 2 h before exposure to 9.5 Gy radiation. Control mice did not receive opaganib or radiation. Animals were then sacrificed on Day 4 or 10, and the small intestines were sectioned and stained with H&E. Representative sections are shown. Panel (A): unirradiated control, Panel (B): vehicle treated at 4 days, Panel (C): opaganib-treated at 4 days, Panel (D): vehicle treated at 10 days and Panel (E): opaganib-treated at 10 days. Villus height is indicated by the brackets and arrows indicate crypt destruction.
Figure 4
Figure 4
Single-dose opaganib improves survival of partially shielded irradiated mice. C57BL/6 mice were orally treated with 0 (▼), 100 (●) or 300 (∎) mg/kg opaganib 4 h before being exposed to 15.25 (Left Panel) or 16.0 (Right Panel) Gy irradiation with 5% bone marrow shielding. Control mice did not receive opaganib or radiation (◊). Mice were monitored for survival for 30 days.
Figure 5
Figure 5
Pre- and post-radiation treatment with multiple-dose opaganib improves survival of partially shielded irradiated mice. C57BL/6 mice were orally treated with 0 (▼), 50 (▲) or 100 (●) mg/kg opaganib 4 h before being exposed to 15.25 (Left Panel) or 16.0 (Right Panel) Gy irradiation with 5% bone marrow shielding. Control mice did not receive opaganib or radiation (◊). Mice were treated with opaganib twice daily at the same dose as their initial treatment for a total of 3 days after radiation. Mice were monitored for survival for 30 days.
Figure 6
Figure 6
Post-radiation treatment with multiple-dose opaganib improves survival of partially shielded irradiated mice. C57BL/6 mice were orally treated with 0 (▼), 50 (▲) or 100 (●) mg/kg opaganib 24 h after being exposed to 15.25 (Left Panel) or 16.0 (Right Panerl) Gy irradiation with 5% bone marrow shielding. Control mice did not receive opaganib or radiation (◊). Mice were treated with opaganib twice daily at the same dose as their initial treatment for a total of 3 days after radiation. Mice were monitored for survival for 30 days.
Figure 7
Figure 7
Survival advantage of opaganib when given after radiation exposure. The percent decrease in mortality observed at the indicated cumulative dose of opaganib given 24 h after 16.0 Gy of radiation across multiple experiments are plotted. A single dose of opaganib was given in the experiment indicated by the open symbol (□), whereas all other data derives from multiple dose treatment given over 2, 3 or 5 days following radiation (∎).
Figure 8
Figure 8
Opaganib sensitizes PAN02 cells to killing by radiation In vitro. Cells were plated and treated with 0 (open symbols) or 10 μM (filled symbols) opaganib and 0 (◯, ●), 5 (△, ▲) or 15 (◆, ◇) Gy of radiation. At the indicated times, viable cell numbers were counted using the trypan blue exclusion assay.
Figure 9
Figure 9
Antitumor efficacy of opaganib and radiation toward pancreatic tumors. C57/BL6 mice were subcutaneously injected with 106 PAN02 cells. When tumors reached 100–150 mm3, animals were randomly assigned into one of four groups (n = 10/group): Vehicle (●); 25 mg/kg opaganib daily (5×/week) (∎); fractionated TBI (1 Gy, 3 times ↑) (▲); or combination of opaganib and TBI (▼). Mice in the combination group were treated with opaganib 2 h prior to radiation.
Figure 10
Figure 10
Antitumor efficacy of opaganib and chemoradiation toward Head and Neck SCC tumors. NCr nu/nu mice were injected subcutaneously with human FaDu tumor cells. When tumors reached 100–150 mm3, mice were randomized and treated with: Vehicle alone (●); opaganib 50 mg/kg/day, 5×/week (∎); fractionated TBI (3 Gy, 3 times ↑) plus cisplatin (2 mg/kg on all TBI days, 2 h pretreatment) (TBI + cisplatin) (▲); or TBI + cisplatin + opaganib (▼). Mice in the combination group were treated with opaganib 2 h prior to radiation. # indicates p < 0.02 for TBI + cisplatin + opaganib compared with TBI + cisplatin.
Figure 11
Figure 11
Model for prevention of GI-ARS and concurrent sensitization of tumor cells to killing by radiation. Levels of S1P and ceramides are represented by red and green slices, respectively. In normal GI tissue, radiation causes an increase in S1P leading to inflammatory tissue damage. Because opaganib treatment reduces the basal level of S1P in the GI tissue, the increase following radiation is insufficient to generate the pathologic inflammatory tissue response. In tumor tissue, the basal level of S1P is higher than normal tissue due to growth factor and/or oncogene driven sphingolipid hydrolysis, and in this case radiation elevates ceramide levels thereby decreasing tumor growth. Treatment of tumors with opaganib restores the resting S1P/ceramide balance and prevents radiation-stimulation of S1P formation, leading to elevation of ceramide sufficient to drive tumor cells into apoptosis.
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