Radiation-induced AGS causes death much faster than BM death (1
). Additionally, since most radiation events are expected to deliver inhomogeneous exposure, many victims will have some marrow shielding and therefore reduced risk of lethal BM syndrome. While the impact of AGS is likely to dominate many situations, to date, no satisfactorily effective mitigation agents have been demonstrated that can rescue or prolong life after near total body radiation-induced AGS.
An appropriate model system is critical for detecting gastrointestinal protection. Since BM LD50/30
is lower than GI LD50/7
), a separation of the effects of these two syndromes must be achieved in a way that allows for severe GI disruption but prevents early BM lethality in order to evaluate the utility of AGS mitigators. This was achieved using our sub-TBI model, which has several advantages: 1)
autologous marrow infusion from spared marrow of one limb avoids allogeneic transplantation and its complications; 2)
the method used to protect marrow is reproducible with little variability; 3)
the limited amount of marrow protected by this technique results in a combined injury that simulates expected clinical scenarios. The sub-TBI model allows us to screen GI mitigation agents efficiently and precisely.
This study demonstrated for the first time that a chemically synthesized FGF-2 analog, FGF-P, can be administered 10 min to 4 hours after irradiation as a mitigator of AGS in mice. A dose at 10 mg/kg (given i.m. daily for 5 days) was active, with a DMF similar to that previously described for human recombinant FGF-2 (DMF ≈ 1.1 to 1.15) (15
). The response of human recombinant FGF-2, a 151aa protein, is modal, with a maximal benefit typically seen at about 0.3 mg/kg/mouse, when given in single or paired doses. Doses over 1 mg/kg i.m. resulted in loss of FGF-2 benefit in several mouse strains (15
). Compared with FGF-2, FGF-P was effective at a relatively high dose, possibly due to its smaller molecular weight (about 2210 Dalton) as compared with ≈46,000 Dalton for FGF-2, which might be cleared much faster. Due to its size and physical characteristics, a longer exposure time for receptor binding is also available to FGF-2 compared with FGF-P. In preliminary studies, response was maximal at 10 mg/kg, with nearly maximal response at 5 mg/kg (data not shown). In this study, the FGF-P administration schedule was selected for relevance to AGS. Obviously, different schedules and doses should be explored to better utilize FGF-P and improve its DMF.
FGF-P is promising for several reasons: 1) it can be chemically synthesized in large quantities with high purity; 2) it has no gross observable toxicity at the maximum dose we employed, 4 g/kg i.m., indicating the therapeutic window is likely to be > 400, a substantial safety factor; 3) the short amino acid sequence based on the FGF-2 receptor binding site lowers risk of autoimmune reaction; and 4) its production is inexpensive and can be stockpiled for long periods as a powder or solution.
An underlying mechanism through which FGF-P mediates its mitigation effect includes enhanced growth of crypt cells (increases number of crypts), and increased proliferation of crypt cells (BrdU staining). In addition, we also measured villi length of duodenum, jejunum, and ileum. 16 Gy sub-TBI reduced villi length compared with normal mice, and treatment with FGF-P better preserved villi length in the duodenum and jejunum. These observations with FGF-P mirror previous observations with native FGF-2 or its analog (17
). Progenitor crypt cells are responsible for replacing small bowel mucosa. The natural replacement rate of villi after minor radiation damage is very brisk (a few days) (26
) and can be enhanced by exogenous addition of FGF-2 (23
) or its analog (24
). Growth factors also enhance NFκB phosphorylation and inhibit apoptosis. This process may preserve some progenitor cells, could help prolong survival and function of terminally differentiated cells in the villi, and thus, could reduce bacterial translocation and absorptive function.
Physical functions of the bowel were better preserved in FGF-P-treated mice. Reduced stool hemoccult score 3.5 days after IR suggests that GI tissue damage is reduced after FGF-P. Stool formation and weight loss were either less severe or of shorter duration in FGF-P-treated animals.
Notably, toxemia is a biomarker of GI integrity following irradiation, in addition to reflecting host immunological defenses. The extent of toxemia in FGF-P-treated mice was less severe than that of controls, indicating that FGF-P better maintained GI integrity and immunological defense.
The pancreas, itself damaged by irradiation, must also aid in physiological and GI recovery through endocrine and exocrine systems (28
). Reduction of plasma insulin at 3.5 days after irradiation might therefore be due to: 1) reduced production of insulin from beta cells; or more likely, 2) down-regulation of insulin secretion due to low circulating glucose caused by reduced food intake or intestinal malabsorption. FGF-P given to irradiated animals both enhanced insulin levels and prevented severe hypoglycemia.
Finally, elevated circulating inflammation molecules, including IL-6, KC, MCP-1, and TNFα are commonly seen in animals and humans with severe side effects from irradiation and seem to predispose them to more severe fibrovascular consequences of irradiation. As in murine models, humans with intrinsic elevation of these cytokines have increased risk of early and late radiation complications. These inflammatory molecules were normalized in FGF-P-treated animals.
While the results of this study are promising, several questions need to be addressed regarding optimal dose and schedule, length of treatment, and effects of supportive care. We will address these questions in future studies.