Ernest L. Jones

A method for the solubilization of a (1->3)-β-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.

Development of a water-soluble, sulfated (1 → 3)-β-d-glucan biological response modifier derived from Saccharomyces cerevisiae

This report describes a method for the solubilization of micro-particulate (1-->3)-beta-D-glucan. Insoluble glucan is dissolved in methyl sulfoxide and urea (8 M) and partially sulfated at 100 degrees. The resulting water-soluble product is called glucan sulfate. The conversion rate is 98%, and the preparation is endotoxin free as determined by the Limulus lysate procedure. Glucan sulfate is composed of 34.06% C, 6.15% H, 50.30% O, 5.69% S and 3.23% N, and has a repeating unit empirical formula of (C6H10O5)8.3 SO3NH4+.4 H2O, suggesting that, on the average, a sulfate group is substituted on every third glucose subunit along the polymer. Molecular weight averages, polydispersity, and intrinsic viscosity were determined by aqueous high-performance size-exclusion chromatography (HPSEC). Two polymer peaks were resolved. Peak 1 (Mw = 1.25 x 10(6) g/mol) represents < 1% of the total polymer mass. Peak 2 (Mw = 1.45 x 10(4) g/mol) comprises > 99% of polymers. 13C NMR spectroscopy confirmed the beta-(1-->3) interchain linkage. In solution, glucan sulfate polymers self-associate in a triple helix. Glucan sulfate stimulates murine bone marrow proliferation following intravenous administration. The ability to prepare a immunologically active, water-soluble (1-->3)-beta-D-glucan preparation will greatly enhance the clinical utility of this class of compounds.

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.

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.

The application of various protic acids in the extraction of (1 → 3)-β-d-glucan from Saccharomyces cerevisiae

Glucans are (1-->3)-beta-linked glucose polymers which have immune-stimulating capability. The extraction of water-insoluble (1-->3)-beta-D-glucan form Saccharomyces cerevisiae employs hydrochloric acid. Hydrochloric acid is difficult to employ in the large-scale pharmaceutical extraction of glucans due to its corrosive nature and toxicity. To address these concerns, we determined whether acetic, formic or phosphoric acid can be substituted for hydrochloric acid in the process for the isolation of (1-->3)-beta-D-glucan. The resulting microparticulate glucans were employed as the starting material for the production of (1-->3)-beta-D-glucan phosphate. 13C NMR analysis of the glucan phosphates derived from the acetic, formic or phosphoric acid-extracted microparticulate glucan show excellent correspondence to hydrochloric acid extracted glucan and laminarin, a (1-->3)-beta-D-glucan standard, indicating that the primary structure is not altered by the acid used for extraction. Glucan phosphate prepared from hydrochloric acid had a Mw of 7.2 x 10(4) g/mol, rmsz of 17.7 nm, of 1.50 and (eta) of 49.0 mL/g. Glucan phosphate prepared from acetic acid had a primary polymer peak with a Mw of 1.4 x 10(6) g/mol, rmsz of 23.6 nm, I of 1.93 and (eta) of 62.4 mL/g. Glucan phosphate prepared from formic acid had a main polymer peak with a Mw of 1.2 x 10(6) g/mol, rmsz 27.1 nm, I of 1.56 and (eta) of 89.0 mL/g. Glucan phosphate prepared from phosphoric acid had a primary polymer peak with a Mw of 6.6 x 10(5) g/mol, rmsz of 32.3 nm, I of 2.70 and (eta) of 91.3 mL/g. These data indicate that the molecular mass, size, polydispersity and intrinsic viscosity of the glucan phosphate obtained is influenced by the pKa of protic acid employed to extract the microparticulate glucan. However, the primary structure and side-chain branching are not substantially altered regardless of the acid employed.

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.

Comparison of the carbohydrate biological response modifiers Krestin, schizophyllan and glucan phosphate by aqueous size exclusion chromatography with in-line argon-ion multi-angle laser light scattering photometry and differential viscometry detectors.

A major barrier to the development, preclinical and clinical application of natural carbohydrate biological response modifiers has been the difficulty involved in accurately characterizing carbohydrate polymers with molecular masses ranging from 10(4) to 10(7) g/mol. Herein, we employed size exclusion chromatography with multi-angle laser light scattering and differential viscometry to compare and contrast structural properties of the biological response modifiers Krestin, schizophyllan and glucan phosphate. Krestin, schizophyllan and glucan phosphate exhibit significant differences in molecular mass moments, molecular mass distribution, polymer sizes, intrinsic viscosity and perhaps their solution behaviour. This knowledge of precise physicochemical data is required for a better understanding of the properties and higher structure of complex carbohydrate biological response modifiers.

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 C57BI/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 < .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 1) resting Kupffer cells are significantly cytotoxic to adenocarcinoma cells at T:E ratios of 1:10 and 1:50 and 2) following a transient inhibition of Kupffer‐cell‐mediated tumoricidal activity, glucan was effective in significantly enhancing the antitumor activity of Kupffer cells.

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.

Protective effect of nonspecific immunostimulation in postsplenectomy sepsis

The enhanced risk of severe sepsis following splenectomy is now well recognized in both adult and pediatric patients. Prophylactic antibiotics and bacterial vaccines have been utilized with limited success to inhibit the high morbidity and mortality. This study reports the use of glucan, a beta-1,3-polyglucose, as a nonspecific immunostimulant for postsplenectomy pneumococcal sepsis. ICR mice were treated with glucan or glucose (5% w/v) following splenectomy or sham operation. Mice were then challenged with 1 X 10(9) Streptococcus pneumoniae intranasally. Glucan significantly increased survival in the splenectomy group (75%) compared to controls (27%). Phagocytic function, as measured by the clearance of 131I-triolein-labeled reticuloendothelial test lipid emulsion, was increased in the glucan group when compared to control glucose animals, both in the presence and absence of pneumococcal infection. Splenectomy alone did not significantly decrease phagocytic function. An increased leukocytosis in response to pneumococcal infection was observed in splenectomized glucan-treated animals. Nonspecific immunostimulation appears to have significant potential as a treatment strategy against postsplenectomy infection.