Development, physicochemical characterization and preclinical efficacy evaluation of a water soluble glucan sulfate derived from Saccharomyces cerevisiae.

This report describes the development, characterization and preclinical efficacy evaluation of water soluble glucan sulfate. Glucan sulfate was derived from insoluble beta-1,3-D-glucan isolated from Saccharomyces cerevisiae. The proposed repeating unit empirical formula of glucan sulfate is [(C6H10O5)5.3H2SO4]n. Two polymer peaks were resolved by aqueous high-performance size exclusion chromatography (HPSEC) with on-line multi-angle laser light scattering (MALLS) photometry and differential viscometry. Peak 1 (MW = 1219697 Da) represents approximately 1% of the total polymers, while peak 2 (MW = 8884 Da) accounts for approximately 99% of polymers. 13C-NMR spectroscopy suggests that glucan sulfate polymer strands may be partially cross-linked. Glucan sulfate (250 mg/kg, i.v.) increased (P less than 0.01) macrophage vascular clearance of 131I-reticuloendothelial emulsion by 42% (P less than 0.01) and in vitro bone marrow proliferation by 46% (P less than 0.05). Glucan sulfate (250 mg/kg, i.v.) increased (P less than 0.05) median survival time of C57B1/6J mice with syngeneic melanoma B16 or sarcoma M5076. In addition, glucan sulfate immunoprophylaxis increased resistance of mice to challenge with Escherichia coli, Candida albicans or Mouse Hepatitis Virus strain A-59. We concluded that: (1) insoluble beta-1,3-D-glucan can be converted to a water soluble sulfated form; (2) glucan sulfate activates macrophages and stimulates bone marrow; (3) glucan sulfate exerts antitumor therapeutic activity, and (4) glucan sulfate immunoprophylaxis will modify the course of experimental infectious disease.

A method for the solubilization of a (1----3)-beta-D-glucan isolated from Saccharomyces cerevisiae.

This report describes a method for the solubilization of a micro-particulate beta-D-glucan. Insoluble glucan is dissolved in methyl sulfoxide and urea (8M) and partially phosphorylated at 100 degrees. The resulting water-soluble product is called glucan phosphate. The conversion rate is 70%, and the preparation is endotoxin free as determined by the Limulus lysate procedure. Glucan phosphate is composed of 34.66% C, 6.29% H, 42.83% O, and 2.23% P and has a repeating-unit empirical formula of (C6H10O5)7.PO3H2, indicating a phosphate group substitution on every seventh glucose subunit. Molecular-weight averages, polydispersity, and intrinsic viscosity were determined by aqueous high-performance size-exclusion chromatography (s.e.c.) with on-line, multi-angle laser light scattering (m.a.l.l.s.) photometry and differential viscometry (d.v.). Two polymer peaks were resolved. Peak 1 (Mw = 3.57 x 10(6) daltons), represents approximately 2% of the total polymers, while peak 2 (Mw = 1.10 x 10(5) daltons) comprises approximately 98% of polymers. 13C- and 31P-n.m.r. spectroscopy confirmed the beta-1,3 interchain linkage and the presence of a phosphate group. In solution, glucan phosphate polymers self-associate in a triple-helical arrangement. The ability to prepare a immunologically active, non-toxic, water-soluble beta-D-glucan preparation will greatly enhance the clinical utility of this class of compounds.

The role of complement in glucan-induced protection against septic shock.

Previous studies from our laboratory have shown that glucan will significantly enhance survival, decrease bacteremia, maintain reticuloendothelial function, and reduce histopathology in a murine model of gram-negative septic shock [1]. The present study was undertaken to evaluate the role of complement in glucan-enhanced protection against septic shock. AKR/J mice, which are congenitally C5-deficient, and ICR/HSD mice that were complement-depleted by treatment with purified cobra venom factor (CVF), were injected IP with glucan (50 mg/kg) on days 5 and 3 prior to IP challenge with 1 X 10(8) E. coli. Survival data indicated that glucan (p less than 0.05) increased survival in both C5-deficient and complement-depleted mice. Glucan prophylaxis resulted in a neutrophilic leukocytosis 8 h following E. coli challenge. However, glucan did not alter bone marrow proliferation. We conclude that, 1) glucan's protective effect on survival is not dependent on complement, 2) complement is not required for glucan-induced neutrophilic leukocytosis in this model, and 3) glucan does not enhance bone marrow proliferation in complement-deficient mice.

Soluble glucan and lymphokine-activated killer (LAK) cells in the therapy of experimental hepatic metastases.

Previous studies have indicated the efficacy of adoptive immunotherapy utilizing recombinant interleukin-2 (rIL-2) and lymphokine-activated killer (LAK) cells in the treatment of advanced neoplastic disease. However, this therapeutic approach is associated with considerable toxicity, primarily due to the systemic administration of rIL-2. The present study was undertaken to determine the efficacy of a newly developed water-soluble glucan, when administered in combination with LAK cells, in the therapy of experimental hepatic metastases. Mice were challenged subcutaneously (1 X 10(4) cells) with reticulum cell sarcoma M5076 on day 0. Therapy was initiated on day 15, when a palpable primary tumor mass and hepatic micrometastases were evident, and continued at 3-day intervals up to day 54. Sarcoma-bearing mice received glucan (250 mg/kg) intravenously, either alone or in combination with LAK cells (1 X 10(7)/mouse). Control mice received 5% (wt/vol) dextrose in water. Glucan-LAK cell therapy significantly suppressed primary tumor growth, inhibited the progression of hepatic metastases and prolonged survival in sarcoma-bearing mice. Splenocytes, incubated with rIL-2 for 72 h, exhibited significant natural killer (NK) cell activity and were cytotoxic to sarcoma cells in vitro. Glucan-LAK cell administration resulted in significant increases in splenic NK cell activity and Kupffer cell-mediated tumoricidal activity. In addition, bone marrow proliferation was enhanced following the co-administration of glucan and LAK cells. Due to its nontoxic nature and immunostimulating properties, soluble glucan may prove to be an attractive biological response modifying agent for utilization in adoptive immunotherapy of advanced neoplastic disease.

Effect of glucan on neutrophil dynamics and immune function in Escherichia coli peritonitis.

Previous studies from our laboratory have demonstrated that glucan, a nonspecific immunomodulator, modifies the course of murine Escherichia coli peritonitis. The protective effect of glucan was mediated, in part, by macrophages. In the present study, leukocyte dynamics in the peritoneal cavity and peripheral blood of glucan-treated mice following E. coli challenge was examined. Additional studies examined in vitro bone marrow proliferation, as well as phagocytosis and intracellular killing of E. coli by neutrophils following glucan administration. ICR/HSD mice were injected ip with glucan (150 mg/kg) or dextrose (5% w/v) on Days 5 and 3 prior to ip challenge with 1 X 10(8) E. coli. Glucan increased (P less than 0.05) total peritoneal neutrophil numbers prior to and following septic challenge. Examination of peripheral blood revealed that ip glucan treatment in E. coli peritonitis significantly (P less than 0.001) increased the number of circulating neutrophils. Additionally, neutrophils from glucan-treated mice showed increased phagocytosis of E. coli in vitro. Glucan therapy also increased bone marrow proliferation. We conclude that (1) glucan enhances peritoneal neutrophil levels, (2) peripheral blood neutrophils are increased following glucan and E. coli, (3) ip glucan increases bone marrow proliferation, and (4) neutrophils from glucan-treated mice showed enhanced phagocytosis of E. coli in vitro. Thus, the beneficial effect of glucan is mediated not only by activated macrophages, but also by the neutrophilic leukocyte.

Pre-clinical safety evaluation of soluble glucan.

Soluble glucan, a beta-1,3-linked glucopyranose biological response modifier, is effective in the therapy of experimental neoplasia, infectious diseases and immune suppression. Currently, soluble glucan is undergoing phase I clinical trials. The present study describes the pre-clinical safety evaluation of soluble glucan in mice, rats, guinea pigs and rabbits. ICR/HSD mice and Harlan Sprague-Dawley rats received a single i.v. injection of soluble glucan in doses ranging from 40 to 1000 mg/kg. Soluble glucan administration did not induce mortality, appearance or behavioral changes in mice or rats. In subsequent studies, mice and guinea pigs were injected i.p. with glucan (250 mg/kg) for 7 consecutive days. ICR/HSD mice gained weight at the same rate as the saline-treated controls. In contrast, guinea pigs receiving i.p. injections of soluble glucan showed a significant (P less than 0.05) 10-13% decrease in weight gain over the 7 day period. No other toxicologic, behavioral or appearance changes were noted. To examine chronic toxicity, soluble glucan was administered twice weekly for a period of 30 or 60 days to ICR/HSD mice in the dose of 40, 200 or 1000 mg/kg. No deaths were observed in any group. Chronic glucan administration did not alter body weight, liver, lung or kidney weight. However, a significant splenomegaly was observed in both the 30 and 60 day study. Histopathologic examination showed no tissue alterations at 40 or 200 mg/kg. However, at 1000 mg/kg a mononuclear infiltrate was observed in the liver. Pyrogenicity testing, employing New Zealand white rabbits, revealed that parenteral glucan administration (5 mg/kg) did not significantly alter body temperature. These data indicate that the systemic administration of soluble glucan, over a wide dose range, does not induce mortality or significant toxicity, an important consideration in preparing soluble glucan for parenteral administration to human populations.

Chemoimmunotherapy of experimental hepatic metastases.

Previous studies from our laboratory have demonstrated that particulate glucan is efficacious in the therapy of a syngeneic murine reticulum cell sarcoma (M5706), which specifically metastasizes from its primary site to the liver. The present study was undertaken to examine the therapeutic efficacy of a newly developed soluble glucan, in combination with cyclophosphamide in the treatment of hepatic metastatic disease. Male C57Bl/6J mice were injected subcutaneously on Day 0 with 1 x 10(4) sarcoma cells. Glucan (200 mg per kg i.v.), cyclophosphamide (45 mg per kg i.p.) or glucan and cyclophosphamide were administered beginning on Day 20, when hepatic metastases were evident, and continued at 3-day intervals up to Day 50. Combined therapy with glucan and cyclophosphamide resulted in reduction of hepatic metastatic lesions on Day 36, compared to control. Survival data revealed that the combination of glucan and cyclophosphamide significantly (p less than 0.001) extended median survival time and the time to 100% mortality in an additive fashion, when compared to either therapy alone. Glucan-cyclophosphamide therapy was also effective in decreasing primary tumor weight to a level that was significantly (p less than 0.05) less than when therapy was initiated. In vitro studies revealed that Kupffer cell tumoricidal activity against sarcoma was increased (p less than 0.05) following glucan and cyclophosphamide. Glucan and cyclophosphamide also enhanced bone marrow proliferation and splenocyte response to mitogens in vitro. Additionally, glucan was observed to exert a direct cytostatic effect on sarcoma in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro tumoricidal activity of resting and glucan-activated Kupffer cells.

Kupffer cells compose 80-90% of fixed tissue macrophages and have been suggested to play an important role in hepatic antitumor resistance. In the present study, the ability of resting and activated Kupffer cells to lyse syngeneic mammary adenocarcinoma BW10232 cells was evaluated. Activated Kupffer cells were isolated from C57Bl/6J mice following single of multiple intravenous (IV) injections of glucan (0.45 mg/mouse), a potent macrophage-activating agent. Mice receiving 5% (w/v) dextrose served as control. Resting Kupffer cells induced significant (P less than .05) 4% and 12% specific lysis of adenocarcinoma cells at target:effector ratios of 1:10 and 1:50, respectively. Kupffer-cell-mediated tumoricidal activity was depressed on day 1 following a single IV injection of glucan. By day 3 postglucan, the antitumor activity of Kupffer cells returned to control levels and was enhanced on days 5 and 10. Following multiple IV injections of glucan on days -5, -3, and -1, Kupffer-cell-mediated cytotoxicity was elevated on days 1 and 4. These observations demonstrate that resting Kupffer cells are significantly cytotoxic to adenocarcinoma cells at T:E ratios of 1:10 and 1:50 and following a transient inhibition of Kupffer-cell-mediated tumoricidal activity, glucan was effective in significantly enhancing the antitumor activity of Kupffer cells.

Enhancement of interleukin-1 and interleukin-2 production by soluble glucan.

Soluble glucan, a beta-1,3-linked polyglucose, is a biologic response modifier effective in the therapy of experimental neoplasia, infectious diseases and immunosuppression. Interleukin-1 (IL-1) and interleukin-2 (IL-2) are endogenous immunomodulators which are essential for effective immune responsiveness. In view of its broad spectrum of immunobiological activity, the ability of glucan to enhance the production of IL-1 and IL-2 was evaluated. Splenic IL-1 and IL-2 secretion as well as plasma IL-1 and IL-2 levels were determined in Sprague-Dawley rats receiving glucan (100 mg/kg, i.p.) at intervals ranging from 12 days to 1 h prior to collection of splenocytes and plasma. Glucan (100 mg/kg) was also injected either s.c., i.p. or i.v. on days -4, -3 and -2 prior to harvesting splenocytes on day 0. Splenic macrophage IL-1 production was initially elevated 12 h following glucan injection and was maintained for a 5 day period. IL-2 secretion by splenic lymphocytes was enhanced 6 h post-glucan and remained elevated for an additional 9 days. Plasma IL-1 activity was elevated 12 h post-injection, while IL-2 activity in plasma was enhanced at 1 h post-glucan. Peak IL-1 and IL-2 activity in plasma occurred 9 and 12 days, respectively, following glucan administration. With regard to route of administration, IV glucan was most effective in inducing lymphokine production. This study demonstrates that: (1) glucan will enhance IL-1 and IL-2 production and (2) elevations in lymphokine production can be maintained up to 12 days post-glucan.

Glucan stimulates production of antitumor cytolytic/cytostatic factor(s) by macrophages.

Glucan, a beta 1,3-linked polyglucose, is an effective macrophage activating and tumor-inhibitory agent. Previous studies indicate that glucan enhances macrophage-mediated tumoricidal activity. The present study was designed to examine the ability of glucan to enhance the production of tumor cell cytotoxic/cytostatic factor(s) designated, because of their macrophage origin, as macrophage cytotoxic factor(s) (MCF). Resting splenic macrophages in culture for 20 h secreted detectable levels of MCF. Coincubation of macrophages with bacterial endotoxin lipopolysaccharide (LPS) resulted in an enhancement of MCF production, compared to resting macrophages. Glucan-activated macrophages were more effective in producing MCF than both resting and LPS-activated macrophages. The MCF was cytotoxic to certain tumor cell lines at high concentrations and cytostatic at lower concentrations. The MCF was not significantly cytotoxic to N3T3 or BC/Sk murine fibroblasts, denoting specificity in response. Loss of MCF activity occurred following coincubation of macrophage culture supernatant with adenocarcinoma cells but not with normal fibroblasts. The MCF activity eluted at 38,000 and 84,000 daltons following column chromatographic analysis, and was heat labile at 100 degrees C but not 56 degrees C. In addition, MCF activity was diminished by protease inhibitors and antisera against tumor necrosis factor. Induction of MCF secretion may be an additional mechanism of glucan-induced antitumor activity.

Comparison of the in vitro cytolytic effect of hepatic, splenic and peritoneal macrophages from glucan-treated mice on sarcoma M5076.

Our previous results have demonstrated that glucan will significantly modify the course of syngeneic murine tumors. Additionally, results denote that glucan increases macrophage-mediated lysis of syngeneic tumor cells. The present study was undertaken to more closely examine the effect of parenteral glucan administration on the tumoricidal activity of hepatic, splenic and peritoneal macrophage populations. C56B1/6J mice were injected intravenously with glucan (0.45 mg/mouse) on days 1,3,6,9,12 and 15. Hepatic, splenic and peritoneal macrophages were isolated on days 8, 12 and 16. The macrophages were co-incubated for 72 h with syngeneic reticulum cell sarcoma M5076 at a target: effector ratio of 1:50. A significant increase in the cytolytic activity of all three macrophage populations was observed. Kupffer cell tumoricidal activity was increased (p less than 0.01) on day 8, but was not significantly different from control on days 12 and 16. Peritoneal exudate macrophages showed increased (p less than .001) tumoricidal activity on days 12 and 16, respectively. Splenic macrophages showed an enhanced (p less than 0.01) increase in lytic activity only on day 16. The present results indicate that: 1) glucan will enhance macrophage-mediated tumoricidal activity of three distinct macrophage populations and 2) a temporal relationship exists between glucan administration and expression of lytic activity by the macrophage populations. These observations further define the mechanism by which glucan exerts its potent anti-tumor activity.

Preparation for hapten help by glucan, muramyl dipeptide, and its L-ala-Glycerol-mycolate derivative.

Previously, we reported that one of the factors that determines whether or not an animal will be prepared for hapten help after priming is the type of adjuvant used. The present work was undertaken, therefore, to determine which of a diverse variety of adjuvants or biological response modifiers would be effective. They included Freund's complete (CFA) and incomplete (FICA) adjuvants, particulate glucan, muramyl dipeptide (MDP), and its L-ala-glycerol-mycolate derivative. Help by the azobenzenearsonate (ABA) hapten was measured as the augmentation of the anti-bovine gamma-globulin (BGG) plaque-forming cell (PFC) response to ABA-BGG of mice that had been hapten-primed with ABA conjugated to ovalbumin (OVA). The results showed that FICA was ineffective. MDP was effective but only if administered with FICA during hapten-priming. MDP-L-ala-glycerol-mycolate was effective without any adjuvant but only within a narrow dose range. Particulate glucan was as effective as CFA in preparing mice for hapten help. As the macrophage is the primary cellular target of those biological response modifiers that were effective, we conclude that it plays an important role in the cellular interaction involved in the mediation of hapten help.

Therapeutic efficacy of glucan in a murine model of hepatic metastatic disease.

Glucan, a particulate beta-1,3-polyglucose immunomodulator, was evaluated for its ability to modify hepatic metastases and survival in mice with reticulum cell sarcoma. Sarcoma M5076 cells were injected subcutaneously (1 X 10(5) cells) into syngeneic C57BL/6J male mice. On Day 20, histopathological studies indicated the presence of hepatic micrometastases. At this time, glucan (0.45 mg per mouse) or dextrose was administered intravenously. Therapy was continued at 3-day intervals up to Day 50. By Day 36 postchallenge, the glucan-treated group, when compared to the control group, showed a marked decrease in hepatic metastases, both grossly and histopathologically. A significant inhibition in the growth of the primary tumor also occurred. Plasma clearance of bromosulfophthalein measured on Day 36, denoted that glucan therapy maintained hepatic parenchymal cell functional integrity, while a 4-fold impairment in bromosulfopthalein removal was observed in control mice. Glucan-treated mice showed a 28% (p less than 0.05) long-term survival. In contrast, control mice showed a 100% mortality by Day 42 postchallenge. Studies to evaluate the mechanism of the anti-metastatic action of glucan indicated that 8 days after glucan administration, isolated hepatic macrophages were significantly more cytotoxic to sarcoma cells in vitro than were normal Kupffer cells. At this time, the cytotoxic activity of peritoneal and splenic macrophages from glucan-treated mice were unaltered. Additionally, co-incubation of particulate glucan with diverse populations of normal or tumor cells in vitro indicated that glucan exerted a direct cytostatic effect on sarcoma and melanoma cells and, in contrast, had a proliferative effect on normal spleen and bone marrow cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Modification of post-operative C. albicans sepsis by glucan immunostimulation.

Glucan, a beta-1,3 polyglucose, was evaluated for its ability to enhance resistance of post-operative mice to experimentally induced C. albicans sepsis. Male C57BL/6J mice were injected i.v. with glucan (0.45 mg/mouse) on days 10,7,4 and 1 prior to midline laparotomy and intravenous challenge with 3 X 10(6) C. albicans. The detrimental effect of surgery on survival following C. albicans infection was manifested by a 47% survival in the non-surgery-infected group in contrast to 20% in the surgery-infected group. Protection against C. albicans was observed in the glucan-treated groups. The glucan-treated non-operated mice manifested 100% survival while the surgery group had a 73% survival. Glucan significantly enhanced macrophage phagocytic function in control and operated mice. Laparotomy alone did not significantly depress macrophage phagocytosis. Histopathological studies revealed that glucan markedly inhibited the renal pathology associated with C. albicans challenge both in the presence and absence of laparotomy. These data indicate that glucan increased survival and reduced renal pathology associated with C. albicans challenge in the post-operative period. These observations suggest that Biologic Response Modifiers such as glucan may be effectively employed in patients who are at risk for post-operative infections.

Immunotherapeutic modification of Escherichia coli--induced experimental peritonitis and bacteremia by glucan.

Previous data from our laboratory have demonstrated that glucan administration significantly alters the course of a variety of experimentally induced infectious diseases. In view of the increasing incidence of gram-negative infections, studies were initiated to evaluate the effect of intraperitoneal glucan therapy on Escherichia coli-induced peritonitis and sepsis. Male ICR/Tex mice were injected intraperitoneally with glucan or dextrose on days 5 and 3 prior to intraperitoneal challenge with 1.0 x 10(8) E. coli. Glucan administration resulted in a significant enhancement of survival. Evaluation of the mechanism of protective action of glucan revealed that both the glucan and dextrose control groups showed an equivalent level of blood-borne E. coli at early periods. At 6 hours after challenge the glucan group showed a significant decrease in blood-borne E. coli. In contrast, the dextrose control group demonstrated progressive bacteremia. A significant depression of phagocytic activity occurred in E. coli-infected mice as compared with control mice that were not exposed to the bacterial challenge. The enhancement in phagocytic function observed in glucan-treated control mice was unaltered in E. coli challenged, glucan-treated mice. The possible importance of hyperfunctional macrophages in reduction of mortality from E. coli sepsis was denoted by methyl palmitate-induced reversal of the glucan hyperfunctional state. Methyl palmitate-treated glucan injected mice were not protected against E. coli infection. These data denote that the intraperitoneal administration of glucan significantly modifies the course of E. coli-induced peritonitis and bacteremia due, in part, to glucan-induced enhancement of macrophage function.