Henry A. Pretus

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.

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.

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.

Application of Aqueous Gel Permeation Chromatography with In-Line Multi-Angle Laser Light Scattering and Differential Viscometry Detectors for the Characterization of Natural Product Carbohydrate Pharmaceuticals

Abstract Natural product complex carbohydrate polymers are currently being developed as prophylactic and/or therapeutic drugs. These water-soluble carbohydrate pharmaceuticals, which are primarily β-1, 3-D-glucan polymers, belong to the class of drugs known as biological response modifiers (BRMs). A major obstacle to the development, understanding and clinical utilization of carbohydrate BRMs has been the difficulty involved in accurately characterizing high molecular weight (MW) carbohydrate polymers. Recent advances in aqueous gel permeation chromatography (GPC), differential viscometry (DV) and multi- significance of these observations are readily apparent. As stated above, the characterization and quality control of natural product carbohydrate BRMs has, in the past, relied heavily on bioassays and conventional GPC. The data presented demonstrate the applicability of high performance GPC with multiple detectors in the analysis of complex carbohydrate BRMs. This technology when combined with other anal...

Inhibition of soybean lipoxygenase by SKF 525-A and metyrapone

To determine whether agents which inhibit cytochrome P-450 enzymes also inhibit lipoxygenase, the effects of metyrapone and SKF 525-A were assessed on soybean lipoxygenase using a spectrophotometric technique which allows for measurement of both the rate and magnitude of product formation. Both SKF 525-A and metyrapone inhibited the rate of product formation and the final amount of product formed in 5 min incubations SKF 525-A was 5 to 5 times more potent than metyrapone, with the IC50 for SKF 525-A 40 microM and for metyrapone between 150 and 200 microM as determined by the total product formation in 5 minutes. Analysis of the reduced product by HPLC confirmed that the substances monitored were those generated by the 15-lipoxygenase enzyme.

Lipoxynoids in the kidney

Abstract There are a variety of non-prostaglandin pathways for conversion of arachidonic acid, including lipoxygenase enzymes and epoxygenase enzymes such as cytochrome P-450. In a manner similar to that in which the cyclooxygenase pathways lead to the prostanoid family, ‘lipoxynoids’ refers to the family of products arising from this alternative group of pathways. Leukotrienes (LTs) are members of the lipoxynoid family arising from the action of 5-lipoxygenase enzymes. In the canine kidney, injections of leukotrienes C4, D4 and E4 into the renal artery produced weak vasodilation at doses of 3–30 ug. Responses to LTC4 and LTD4 were similar and greater than responses to LTE4, and responses were not different in animals which had received ibuprofen to inhibit prostaglandin synthesis. In contrast, these leukotrienes were potent vasoconstrictors of the mesenteric vascular bed in these same animals at doses of 0.01–0.3 ug. The order of potency was LTD4 LTC4 LTE4. Effects of these LTs were not changed in the presence of ibuprofen. Responses to LTC4 and LTD4, but not LTE4 were diminished approximately 50% after administration of FPL-55712 (2 mg/kg). Neither LTB4 nor 5-HETE produced any change in renal or mesenteric blood flow at doses up to 30 ug. However, indirect evidence has been obtained suggesting that an endogenous lipoxynoid pathway can be activated in the canine kidney which results in the formation of a vasoconstrictor product. Injections of 1–4 mg AA into the renal artery of water-replete dogs leads to vasodilation which can be blocked by inhibitors of cyclooxygenase enzymes. However, when dogs were water deprived for 16–20 hours before the experiment, biphasic changes in renal blood flow were found. Ibuprofen blocked the vasodilator phase of the response but neither ibuprofen or the thromboxane synthesis inhibitor OKY-1581 had any inhibitory effect on the constrictor phase. The constrictor phase was blocked only following administration of ETYA or BW-755C, suggesting that the metabolites responsible for the constriction were lipoxynoids. Since LTs produce renal vasodilation, it appears that the pathway involved is not the 5-lipoxygenase system. These data suggest that other lipoxynoid pathways (e.g. 12-lipoxygenase, 15-lipoxygenase or cytochrome p-450) may play a role in the renal response to water deprivation. At present, however, it may not be possible to distinguish between these possible pathways in vivo .

Inhibition of lipoxygenase enzyme in vitro by the cytochrome P-450 inhibitors metyrapone and SKF-525-A

Abstract It has recently been demonstrated that agents which block lipoxygenase enzymes (i.e. nordihydroguaiaretic and eicosatetraynoic acid) also block cytochrome P-450 in some in vitro preparations. In some cases, therefore, results which were based on these lipoxygenase inhibitors could have been produced by the inhibition of cytochrome P-450. We therefore sought a way to distinguish between effects produced by metabolites of arachidonic acid generated by lipoxygenase enzymes and those produced by metabolites of the cytochrome P-450 system. seemed that a straightforward approach might be to administer drugs that inhibit the cytochrome P-450 system first, and then examine effects of lipoxygenase inhibitors. However, it then became necessary to establish the effect of inhibitors of the cytochrome P-450 system on lipoxygenase enzymes. Initial work was carried out using crystalline soybean lipoxygenase enzyme to avoid the complexities involved in isolating enzymes from biological tissues. Enzyme activity was assessed spectrophotometrically by measuring the increase in absorbance at 240 nm produced by the formation of a conjugated triene (15-HETE) from arachidonic acid. Effects of the two most commonly used cytochrome P-450 antagonists, metyrapone and SKF-525A, were determined by Lineweaver-Burke analysis. One major difficulty was that arachidonic acid is soluble in aqueous media at Ph 8 whereas both SKF-525-A and metyrapone are soluble at pH 7. In order to maintain all components in solution at the same time, experiments were carried out at pH 7.35 in the presence of 5% dimethylsulfoxide. There was no observable difference in the activity of 15-lipoxygenase at pH 9 in the absence of DMSO and that observed at pH 7.35 in the presence of DMSO. Studies were carried out in 2 ml quartz spectrophotometer cuvettes. For each experiment, two cuvettes were prepared containing arachidonic acid, DMSO (0.1 ml), SKF-525-A or metyrapone, and Tris buffer (pH 7.35). The cuvettes were placed in a Beckman DB/G spectrophotometer. One cuvette was used as reference and to the other was added 3ug (0.1 ml) of a solution of crystalline soybean lipoxygenase (Sigma). The change in absorbance at 240 nm was recorded and reflected product formation. Concentrations of arachidonic acid ranged from 10–50 uM, each tube contained either 0–50 uM SKF-525-A or 0–200 uM metyrapone. Results show that both SKF-525-A and metyrapone inhibited soybean lipoxygenase. Lineweaver-Burke analysis indicated that both agents acted in uncompetitive fashion. Of particular importance is that these concentrations of SKF-525-A and metyrapone are similar to those routinely used to block cytochrome P-450. From these results, it appears that experiments in which SKF-525-A and metyrapone were employed may need to be reevaluated. Work is underway using platelet-derived 12-lipoxygenase.