Myra L. Patchen

Therapeutic Use of Recombinant Human G-CSF (rhG-CSF) in a Canine Model of Sublethal and Lethal Whole-body Irradiation

The short biologic half-life of the peripheral neutrophil (PMN) requires an active granulopoietic response to replenish functional PMNs and to maintain a competent host defence in irradiated animals. Recombinant human G-CSF (rhG-CSF) was studied for its ability to modulate haemopoiesis in normal dogs as well as to decrease therapeutically the severity and duration of neutropenia in sublethally and lethally irradiated dogs. For the normal dog, subcutaneous administration of rhG-CSF induced neutrophilia within hours after the first injection; total PMNs continued to increase (with plateau phases) to mean peak values of 1000 per cent of baseline at the end of the treatment period (12-14 days). Bone-marrow-derived granulocyte-macrophage colony-forming cells (GM-CFC) increased significantly during treatment. For a sublethal 200 cGy dose, treatment with rhG-CSF for 14 consecutive days decreased the severity and shortened the duration of neutropenia and thrombocytopenia. The radiation-induced lethality of 60 per cent after a dose of 350 cGy was associated with marrow-derived GM-CFC survival of 1 per cent. Treatment with rhG-CSF markedly reduced the lethality associated with exposure to 350 cGy of radiation to zero. White blood cell (WBC) and platelet recovery kinetics were correlated with degree of marrow damage. The rhG-CSF reduced the severity and duration of neutropenia. Control animals required antibiotic therapy (WBC less than 1000 mm3) for a total of 16 days versus 3 days for rhG-CSF-treated dogs. The duration of thrombocytopenia was reduced, although the severity of depletion was unchanged with treatment. These data indicate that in the lethally irradiated dog, effective cytokine therapy with rhG-CSF will increase survival through the induction of earlier recovery of neutrophils and platelets.

Glucan: mechanisms involved in its "radioprotective" effect.

It has generally been accepted that most biologically derived agents that are radioprotective in the hemopoietic‐syndrome dose range (eg. endotoxin, Bacillus Calmette Guerin, Corynebacterium parvum, etc) exert their beneficial properties by enhancing hemopoietic recovery and hence, by regenerating the hosts ability to resist life‐threatening opportunistic infections. However, using glucan as a hemopoietic stimulant/radi‐oprotectant, we have demonstrated that host resistance to opportunistic infection is enhanced in these mice even prior to the detection of significant hemopoietic regeneration. This early enhanced resistance to microbial invasion in glucan‐treated irradiated mice could be correlated with enhanced and/or prolonged macrophage (but not granulocyte) function. These results suggest that early after irradiation glucan may mediate its radioprotection by enhancing resistance to microbial invasion via mechanisms not necessarily predicated on hemopoietic recovery. In addition, preliminary evidence suggests that glucan can also function as an effective free‐radical scavenger. Because macrophages have been shown to selectively phagocytize and sequester glucan, the possibility that these specific cells may be protected by virtue of glucans scavenging ability is also suggested.

16,16-Dimethyl prostaglandin E2 increases survival in mice following irradiation

16,16-Dimethyl prostaglandin E2 (DiPGE2), a stable analog of PGE2, increases the LD50/30 survival in CD2F1 male mice when given prior to ionizing radiation. Subcutaneous administration of 40 micrograms of DiPGE2 30 min prior to 60Co gamma irradiation extends the LD50/30 from 9.39 Gy in the control animals to 16.14 Gy in DiPGE2 treated, with a dose reduction factor of 1.72 [95% confidence limits: 1.62, 1.82]. The degree of protection is dependent on both the time of administration and the dose of the prostaglandin. Ten micrograms administered 5 min prior to receiving a lethal dose of 10 Gy provides 90% survival but only 10% survival if administered 30 min prior to irradiation. Experiments to determine the in vivo concentration of DiPGE2 in organs postinjection show increased levels over time, but these are not correlated with protection. At 30 min after injection, as much as 80% of the DiPGE2 present in the spleen and plasma is unmetabolized. These results suggest that the protection results from the physiologic action of DiPGE2 rather than direct in vivo detoxification of radicals.

Stimulated hemopoiesis and enhanced survival following glucan treatment in sublethally and lethally irradiated mice

Hemopoietic effects of the reticuloendothelial agent glucan were assayed in normal mice and in mice hemopoietically depleted by exposure to 60Co radiation. In normal mice, glucan administration increased the content of bone marrow and splenic transplantable pluripotent hemopoietic stem cells (CFU-s), committed granulocyte-macrophage progenitor cells (GM-CFC), and pure macrophage progenitor cells (M-CFC). Erythroid progenitor cells (CFU-e) were increased only in the spleen. In sublethally irradiated mice (650 rads), glucan increased the number of endogenous pluripotent hemopoietic stem cells (E-CFU) when administered either before or after irradiation. The most pronounced effects were observed when glucan was administered 1 day before, 1 h before, or 1 h after irradiation. In addition, the administration of glucan before lethal irradiation (900 rads) enhanced survival. The most significant results were seen when glucan was administered 1 day prior to irradiation. The possibility of using agents such as glucan to enhance hemopoietic reconstitution and prevent septicemia following chemotherapy and/or radiotherapy is discussed.

Combined Modality Radioprotection: The Use of Glucan and Selenium with WR-2721

Glucan, WR-2721, and selenium, three agents with distinct radioprotective mechanisms, were evaluated in C3H/HeN mice for survival-enhancing and hemopoietic-regenerating effects when administered alone or in combinations before exposure to 60Co radiation. At LD50/30 radiation doses (radiation doses lethal for 50% of mice within 30 days postexposure), dose reduction factors of 1.21, 1.02, 1.37, 1.51, and 1.66 were obtained following glucan (75 mg/kg i.v., -20 hr), selenium (0.8 mg/kg, i.p., -20 hr), WR-2721 (200 mg/kg, i.p., -30 min), glucan + WR-2721, and glucan + selenium + WR-2721 treatments, respectively. All treatments increased numbers of hemopoietic stem cells as measured by the day 12 endogenous spleen colony-forming unit (E-CFU) assay; the most significant E-CFU effects, however, were observed following glucan + WR-2721 and glucan + selenium + WR-2721 treatments. Combined modality treatments were also more effective than single-agent treatments at accelerating bone marrow and splenic granulocyte-macrophage colony-forming cell (GM-CFC) regeneration. These results demonstrate the value of multiple-agent radioprotectants.

Postirradiation treatment with granulocyte colony-stimulating factor and preirradiation WR-2721 administration synergize to enhance hemopoietic reconstitution and increase survival

These studies tested whether WR-2721 could be used to protect hemopoietic stem cells, which after irradiation could be stimulated by granulocyte colony-stimulating factor (G-CSF) to proliferate and reconstitute the hemopoietic system. Female C3H/HeN mice were administered WR-2721 (4 mg/mouse, i.p.) 30 min before 60Co irradiation and G-CSF (2.5 micrograms/mouse/day, s.c.) from days 1-16 after irradiation. In survival studies, saline, G-CSF, WR-2721, and WR-2721 + G-CSF treatments resulted in LD50/30 values of 7.85 Gy, 8.30 Gy, 11.30 Gy, and 12.85 Gy, respectively. At these LD50/30 values, the dose reduction factor (DRF) of 1.64 obtained in combination-treated mice was more than additive between the DRFs of G-CSF-treated mice (1.06) and WR-2721-treated mice (1.44). Bone marrow and splenic multipotent hemopoietic stem cell (CFU-s) and granulocyte-macrophage progenitor cell (GM-CFC) recoveries were also accelerated most in mice treated with WR-2721 + G-CSF. In addition, mice treated with WR-2721 + G-CSF exhibited the most accelerated peripheral blood white cell, platelet, and red cell recoveries. These studies (a) demonstrate that therapeutically administered G-CSF accelerates hemopoietic reconstitution from WR-2721-protected stem and progenitor cells, increasing the survival-enhancing effects of WR-2721 and (b) suggest that classic radioprotectants and recombinant hemopoietic growth factors can be used in combination to reduce risks associated with myelosuppression induced by radiation or radiomimetic drugs.

Enhanced Hematopoietic Recovery in Irradiated Mice Pretreated with Interleukin-1 (IL-1)

Data in this report compare the number of colony-forming cells (CFC) in bone marrow from irradiated and pre-irradiated C57Bl/6J mice injected with saline or recombinant interleukin-1-alpha (rIL-1). Eight to 12 days after sublethal or lethal irradiation, there were more CFU-E (colony-forming units-erythroid), BFU-E (burst-forming units erythroid), GM-CFC (granulocyte-macrophage colony-forming cells), and day 8 CFU-S (colony-forming units-spleen) in bone marrow from rIL-1 injected mice than from saline injected mice. Prior to irradiation, there was no increase in number of CFC in bone marrow from rIL-1 injected mice. However, as determined by sensitivity to hydroxyurea, rIL-1 injection stimulated GM-CFC into cell cycle. These results demonstrate that rIL-1 injection increases the number of CFC that survive in irradiated mice and may be a consequence of the stimulation of CFC into cell cycle prior to irradiation.

Comparative effects of particulate and soluble glucan on macrophages of C3H/HeN and C3H/HeJ mice

In order to compare both the actions of soluble glucan (glucan-F) and particulate glucan (glucan-P) on macrophages and the responsiveness of macrophages from C3H/HeJ and C3H/HeN mice to these immunomodulators, interleukin-1 (IL-1) levels, phagocytosis and superoxide production were monitored after an in vitro exposure to glucan-F or glucan-P. A 2 or 20 h exposure to either glucan preparation decreased the ability of both C3H/HeJ and C3H/HeN macrophages to ingest zymosan. In contrast, glucan-P, but not glucan-F, decreased (after a 20 h exposure) the uptake of both IgG opsonized erythrocytes and latex beads. Furthermore, glucan-P, but not glucan-F was as effective as zymosan (after a 1 h exposure) in inducing superoxide release by macrophages isolated from both C3H/HeN and C3H/HeJ mice. While the effects of glucan-P on PMA-induced superoxide release and IL-1 levels were similar in macrophages from C3H/HeJ and C3H/HeN mice, glucan-F was ineffective at enhancing PMA-induced superoxide release or increasing IL-1 levels in C3H/HeJ mice. Thus (1) the effects of glucan-P on phagocytosis of opsonized erythrocytes and latex beads are not mimicked by glucan-F and (2) while macrophages from C3H/HeJ mice respond normally (as compared with C3H/HeN macrophages) to glucan-P, they are hyporesponders to glucan-F. These findings indicate that the activation of macrophages by glucan-P involves different (or additional) pathways from those activated by glucan-F.

Postirradiation glucan administration enhances the radioprotective effects of WR-2721

Based on murine survival studies, endogenous hemopoietic spleen colony formation (E-CFU), and recovery of bone marrow and splenic granulocyte-macrophage colony-forming cells (GM-CFC), it was demonstrated that the postirradiation administration of glucan, an immunomodulator and hemopoietic stimulant, enhances the radioprotective effects of WR-2721. LD50/30 dose reduction factors for mice treated with WR-2721 (200 mg/kg approximately 30 min before irradiation), glucan (250 mg/kg approximately 1 h after irradiation), or both agents were 1.37, 1.08, and 1.52, respectively. Enhanced survival in mice treated with both agents appeared to be due in part to glucans ability to accelerate hemopoietic regeneration from stem cells initially protected from radiation-induced lethality by WR-2721. Following a 10-Gy radiation exposure, E-CFU numbers in mice treated with saline, WR-2721, glucan, or both WR-2721 and glucan were 0.05 +/- 0.03, 6.70 +/- 1.05, 0.95 +/- 0.24, and 33.90 +/- 2.96, respectively. Similarly, bone marrow and splenic GM-CFC numbers were greater in mice treated with both WR-2721 and glucan than in mice treated with either agent alone. These results demonstrated at least additive radioprotective effects when mice were given WR-2721 prior to irradiation and glucan following irradiation. These effects appeared to depend on the sequential cell protection mediated by WR-2721 and hemopoietic repopulation mediated by glucan.

A comparative evaluation of particulate and soluble glucan in an endotoxin model

Particulate glucan (P) but not soluble glucan (F) has been shown to sensitize rats to endotoxins. This phenomenon is believed to be mediated by the reticuloendothelial system (RES). The effect of glucan-P and -F on the RES, and the response of glucan-treated rats to nonlethal doses of endotoxin were investigated. Rats were injected for 5 days with 10 mg/kg of glucan-P, -F or saline. Three days later rats were either (1) injected with colloidal carbon for clearance studies, (2) sacrificed for organ histology and determination of serum glucose, plasma thromboxane (Tx) B2, and plasma 6-keto-prostaglandin (PG) F1 alpha concentrations, or (3) challenged with a nonlethal dose of endotoxin. The latter were further subdivided into groups for either 30-day survival or for sacrifice at 30 min or 4 h post-endotoxin infusion to obtain blood samples for glucose, TxB2, and 6-keto-PGE1 alpha determinations. Glucan-P induced hepatosplenomegaly and granulomatous changes within the liver and spleen. The carbon clearance halftime was markedly decreased in these animals. In glucan-P-treated rats challenged with endotoxin, elevated concentrations of both plasma prostanoids were observed as well as alterations in serum glucose levels. These changes were less pronounced in glucan-F- or saline- treated rats. Following endotoxin challenge, only 40% of glucan-P-treated rats survived 30 days whereas 100% of both the glucan-F and saline-treated rats survived. We conclude that glucan-P, in contrast to glucan-F, significantly heightens RES function and that this effect likely accounts for the endotoxin sensitivity.