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Chapter 14. Biological Response Modification by β-D-Glucans
Chapter 14.
Blologlcal Response Modification by R-D-Glucans
Robert C. Goldman Abbott Laboratories, Pharmaceutical Research Division D-47M/AP9A 100 Abbott Park Rd., Abbott Park, IL 60064-3500
introduction - The mammalian immune defense system is a complex and highly
evolved assemblage, functioning to defend the organism from constant microbial attack from without, and as well disease from within in the form of emerging tumor cells. Although the immune system in its normal state is quite successful in performing its protective role, the slightest decline in functional capacity can predispose the host to infection, and/or lowering of anti-tumor immune surveillance. Furthermore, specific patient populations with normal immune system function are still at risk for infection in certain clinical settings. Thus exogenous stimulation of immune system function is a logical clinical approach to positive intervention. One approach uses endogenous molecules of the immune system (e.g. various cytokines), while another uses exogenous elements, usually of microbial origin. The current report will focus on fungal glucans and their potential for clinical development. b k a r o u d - Zymosan, a cell wall preparation derived from Saccharomyces cerevisiae, has diverse physiological effects, many of which can be beneficial, Cell wall glucan was implicated as the active component in zymosan (l), and subsequent studies revealed that 0-glucans in general did evoke many of the beneficial effects, including stimulation of resistance to microbial infection and activation of anti-tumor responses. Although no glucan-derived products are yet approved for clinical use in the United States, glucans are used elsewhere in specific clinical settings. The structure, biological activity, mode of action, preclinical and clinical data pertaining to glucans will be reviewed.
- Natural product glucans consist of a linear backbone of 0-1,3 linked D-glucopyranosyl groups with varying degrees of branching from the C6 position. Most repolted degrees of branching, i.e., the number of brancheslmain chain residues (DB), should be considered average values with the possibility for heterogeneity in branch distribution as was indicated for glucan isolated from Volvariella (2). Branches are usually only a single glucose residue, although more than one glucose unit may be present in some glucans (3,4), and see PGG-glucan and lentinan below. The repeating unit of natural product glucans with varying branching frequencies include: Alcaligenes (curdlan), Saccharomyces (5), Omphalia OR-OL-~C,Aspergillus (6), Agrocybe (7) and Euglena (paramylon), (DBsO), Omphalia OL-2-111, 1 (DB=1/24), Volvariella, 2 (DBP1/5.5), 1 n 23, 2 n = 5-51a = Fusarium, 3. (DB=1/5), Cryproporus, 9 (DB=1/4), 3s = 3.5* 2v Amanita glucan, 5, (DB=2/7), Grifola (grifolan), L n = l , 8 n=0.5 Omphalia OL-2-1, Schizophyllum (schizophyllan, Schizofiran), and Sclerotinia (scleroglucan), fi (DB=1/3), Omphalia OL-2-11, 2 (DB=1/2), and OL-2, (DB=2/3) glucans. Branching frequency can modulate glucan structure and biological activities (see below). 49
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The three dimensional structures of lentinan (a), curdlan (9) and 6, schizophyllan, (1 0) were elucidated by X-ray diffraction studies. Triple helical, parallel strands of glucan are hydrogen bonded via the C2 hydroxyls, with higher order structures arising via hydrogen bonding between C4 and C6 hydroxyls. Since the C6 hydroxyls are on the external surface of the triple helix, branches would project away from the helix surface and likely modulate subtle aspects of glucan structure and biological activity. A stereo view of the glucan triple helix is shown (Figure 1). RGlucans can also adopt single chain and single helix forms, as assessed by NMR spectroscopy (11,12). Linear rods, hairpins, circular, globular, and other structures were identified by electron microscopy depending on the glucan sample and mode of preparation (13,14).
Figure 1 . Stereo view of the glucan triple helix.
Early in evolution defense systems evolved to detect and rapidly respond to fungal infection. R-Glucan, a distinguishing component from fungal cells walls, is thus recognized and a defense response initiated. Data are available for specific receptor recognition of glucans in mammalian and non-mammaliansystems, resulting in signal transduction events leading to regulated defense responses.
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L b n M a ~ n m d h The unit glucan structure binding to a plant glucan receptor was determined, and solubilization and identification by photolabeling of the receptor were reported (15-17). A specific heptasaccharide structure is recognized by the receptor (Kd in the nanomolar range), and elicits plant defense responses. Specific glucan binding proteins also exist in fish and invertebrates, and function to trigger defense systems (18-21). A trimer was the minimal structure interacting with the receptor in salmon, and the intact non-reducing terminus was required (22). The crayfish glucan binding protein may have homology to the glucan binding site of Rglucanase (23).
Mammalian - Complement receptor 3 (CR3)and other receptors are implicated in the
response of mammalian systems to glucans. CR3 is an integrin receptor known to bind several ligands, and apparently 8-glucans as well (24). A role for CR3 in binding glucan
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is supported by the following: 1) antibody against CR3 blocked responses to glucan (25-27), 2) neutrophils from patients with a deficiency of CR3 were unresponsive to Oglucan (26), and 3) CR3 was isolated from neutrophils by affinity chromatography on Oglucan-Sepharose (26). However, CR3 affinity purified on iC3bSepharose did not bind zymosan and derived glucan in a solid phase assay (28). In addition, stimulation of CR3 with glucan up regulates CR3 (29), and stimulates NK cell cytotoxic activity (24,27,30) presumably via iC3b coated on target cells. Although CR3 plays a role in glucan induced synthesis of monocyte platelet-activating factor (PAF), the actual receptor interacting with glucan was considered to be distinct from CR3 (31). Glucan interaction with CR3 can alter PKC activity, and in the presence of vanadate, can lead to PLA2 activation (33). In addition, mouse peritoneal macrophages have a receptor which reversibly bound aminated O-1,3 glucan, resulting in glucan internalization (32). An anti-idiotype (anti-Id) antibody, prepared using a monoclonal antibody against the B-1,3 glucan laminarin coupled to protein (34), was reported to interact with a O-glucan receptor on monocytes. lodinated anti-Id bound to U937 cells, and reacted with proteins of molecular weights 180 and 160 kDa (35) that consisted of subunits (36) which were differentially regulated (37). A heptasaccharideof undefined structure may represent the unit structure interacting with this receptor (38). N K cell surface proteins which bound yeast wall glucans were also identified in a separate study (39). Corticosteroid treatment of human monocytes increased subsequent phagocytosis of glucan particles as well as glucan stimulated secretion of 0-N-acetylglucosaminidase (40), presumably via modulation of glucan receptors. Retinoic acid and 1,25dihydroxyvitamin D3 also induced 0-1,3 glucan receptors on P388D1 cells (41). In addition, crosslinking of receptors may influence cellular responses to glucans (42). JIFRIVATIVFS
0F GLUCANS
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o w d G l u u - Glucan physiochemical properties and biological activity can be modulated by chemical modification. Insoluble yeast glucan was converted to water soluble glucan sulfate, 8, (43,44) yielding material with repeating unit empirical formulas of (CgH1005)8-3 S03NH4 and (C6H1005)5*3H2S04. These modified glucans still formed triple helices in solution, and were capable of stimulating bone marrow proliferation and resistance to = Or microbial challenge in mouse models. Soluble, triple helical glucan phosphate with formula B (C6H1005)7.P03H2,10, was also prepared -?' 9 (45,46). Although the extent of substitutions along 0 the 0-1,3 backbone was determined, no experimental data were given as to the precise -p-o - 1Q positions of the sulfate or phosphate groups. Both 0modified glucans contained few if any branches (5). No significant toxicity was observed with glucan phosphate, which is active in promoting wound healing, stimulating resistance to microbial infection, hematopoesis, anti-tumor responses and protection from irradiation (46). O-1,3-Glucans with varying degrees of branching (DB=O, 1i3 and 1/2 see above) were sulfated by reaction with chlorosulfonic acid-pyridine and converted to the sodium salt for analysis of bioactivity (47). Degree of substitution altered the effects of glucans on plasma clotting, complement activation, macrophage stimulation, and mitogenic activity. In addition, the biological effects of a branched glucan were altered by substitution with carboxymethyl groups, suggesting that receptors recognized specific structural features of native glucan (48).
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Glucan-Drua C o n i u u - Glucan conjugation to cytotoxic drugs modulated toxicity while maintaining the beneficial effects of glucans. Carboxymethylated yeast O-1,3 glucan was coupled to 143-D-arabinofuranosylcytosine(araC) (49) by activating the polysaccharide carboxyl group with isobutylchloroformate and formation of a peptide
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bond via the 4-NH2 group of araC yielding 11. This conjugate possessed enhanced anti-leukemic activity, and maintained the immunomodulatory activity of the glucan. Mitomycin C was also conjugated to carboxymethylated, linear O-1,3 glucan from Alcaligenes in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide(50) yielding 12. The conjugate was active against P388 leukemia, and sarcoma 180 solid tumors in mice. Mitomycin C conjugated 0 HO to carboxymethylated schizophyllan (DB=1/3) was similarly prepared (51). Carboxymethylation was 1? extensive (degree of substitution of 0.87), occurring predominantly at the C4 or C6 hydroxyls. The conjugate was active against sarcoma 180 Nsolid tumors in mice, and 0 caused less reduction of circulating leukocyte numbers compared to mitomycin C alone.
LN VlTROAND IN VlVO RFSPONSFS TO GI UCANS O-glucans affect immune system functions in vitro including NK cell cytotoxicity (24,27,30,39), CR3 receptor up-regulation (29), production of cytokines (52-57), PLA2 (33), PGE2 (56), arachidonic acid (58),and wound healing factors (57). The relationship between the induction of these and other factors can influence immune system response to bacterial challenge (59). ln vivo effects of glucans include:
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ulation of Resistance to Microbial Challenae - High molecular weight triple helical scleroglucan, 6, increased resistance of mice to challenge with Staphylococcus aureus, Candida albicans and mouse hepatitis virus, with little toxicity (60). Glucan phosphate, lQ,was also active in several animal models (46). The N,Ndimethy laminoethylderivative of paramylon (DB=O) stimulated resistance of mice to challenge with R = H or various microorganisms, whereas carboxymethyl paramylon and paramylon sulfate were less I\C--N& effective (61). Yeast derived 0-1,3-glucan also @ ] . :R acted synergistically with antibiotics via enhanced IL-1 secretion by macrophages, and enhanced OR bone marrow proliferation (62). Soluble, aminated &1,3 glucan, J& modulated cytokine production and protected mice against otherwise lethal bacterial infections (63). Although timing of glucan administration was critical in most studies (also see below for PGG-glucan) long term protection against bacterial challenge was induced in rats (64).
[
ion of HematoDoesk - Glucans also enhanced hematopoesis in animal models, possibly via induction of colony stimulating factors. Scleroglucan, & enhanced bone marrow proliferation when administered to mice (60). Soluble glucan alone, and in combination with human granulocyte colony-stimulating factor enhanced survival and hematopoesis in irradiated mice (65,66). Glucan also abrogated the suppressive effects of pefloxacin on hematopoesis following irradiation (67). Soluble yeast-derived glucan also stimulated hematopoesis with increases in serum levels of granulocyte-macrophage colony stimulating activity (68). Chemically modified glucans (glucan sulfate, g, and glucan phosphate 1p) also stimulated murine bone marrow proliferation following intravenous administration (43,46) in mice.
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on of Anti-Tumor ResDo[lSBS - When examined, the anti-tumor activity of glucans appears due to immune system stimulation as opposed to direct activity against tumor cells. Induction of cytokines, and possible activation of cytotoxic cells, are involved (see below). Scleroglucan, 5, (DB=1/3) increased survival of mice challenged with syngeneic lymphoma, melanoma or adenocarcinoma (60), and inhibited pulmonary metastasis in mice when administered orally or intraperitoneally (69). Glucan also reduced mitomycin C toxicity and promoted an anti-tumor response in a mouse model (51). Branched O-glucans of MW 10,000 and 20,000, were active against Sarcoma 180 in mice, while a related glucan of MW 6000 was inactive, suggesting molecular weight may influence glucan activity in specific systems (70). indicated that tumor Investigation of the mode of action of aminated glucan, regression was stimulated via vascular damage to tumor blood flow, and necrosis via functional T cells and TNF (71). A series of branched glucans isolated from Omphalia (DB=2/3) alone or in were tested for anti-tumor activity in the mouse. OL-2, combination with 5-fluorouracil had activity against solid tumor and ascites forms (72). In addition, derivatives of OL-2 with lesser degrees of branching (1,4, 2) showed potent anti-tumor activity against the solid form of sarcoma 180 mice (73). Another branched glucan isolated from Arnanita, & (DBs217) exhibited significant anti-tumor activity against Sarcoma 180 in mice alone and in combination with mitomycin C (3). Carboxymethylation of the linear glucan prepared from Agrocybe yielded a product with activity against the solid form of Sarcoma 180 in mice (7).
u, a,
of C v w - Cytokine induction by glucans in in vivo animal models was reported in several studies. Administration of OL-2, 6, to mice induced expression of cytokines, including several interleukins, colony stimulating factors, and IL-1 receptor antagonist (IL-1ra) mRNA's (74,75). Schizophyllan, 6, induced IL-1 alpha mRNA comparable to that by OL-2, a, but a weaker level of IL-1 beta mRNA, and did not induce IL-1ra (75). Glucan also acted synergistically with antibiotics via enhanced IL-1 secretion by macrophages, and enhanced bone marrow proliferation (62). Scleroglucan, 5, (DB=1/3) enhanced IL-1 production (76), soluble aminated glucan modulated cytokine production and enhanced IL-1 and TNF levels (63), and paramylon (DB=O) induced IL-1 and IL-6 (77), when administered to mice. O - Glucans also ~ enhanced wound healing via modulation of W of the immune system (46,57,78). The strength of healing surgical incisions was increased by I.V. or topical administration of glucan in a rat surgical model, apparently via enhanced macrophage function and increased cross-linking of collagen (57). Glucan also increased the wound healing rate in a mouse second degree burn recovery model, presumably via alteration of macrophage function (78).
.. Admlnlstration
- Several studies have demonstrated biological activity of glucans administered orally to animals. Orally administered glucan, extracted from Candida albicans (Glucanil, Gluimmun), increased resistance to challenge with Candida albicans or Staphylococcus aureus in a mouse model (79). Glucan increased polymorphonuclear leukocytes and peripheral monocytes levels, and the in vitro candicidal activity of recovered alveolar macrophages and spleen cells. Scleroglucan, 6, (DB-113) increased the number of alveolar macrophages, enhanced their lysosomal enzyme activity, phagocytic activity, and IL-1 production, presumably via effects on Peyer's patch cells (80). The proliferative response of Peyer's patch cells to B and T-cell mitogens was also enhanced (81). Scleroglucan, 5, (DB=1/3) inhibited pulmonary metastasis in mice when administered orally or intraperitoneally (69). ies of Glucan via Oral
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.
- Glucan in combination with zinc and pig Affects in Other Rtalpaiimmunoglobulin reduced the number of migrating ascarid larvae in the lungs of pigs (82). Glucan isolated from S. cerevisiae suppressed lesions caused by Leishmania in
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mice (83), and acted as an adjuvant during immunization with Leishmania (84) and Trypanosoma (85). Semisoluble I31,3-carboxymethylglucan increased survival in a mouse model of acute massive hemorrhage (86). Branched glucan decreased the cumulative incidence of diabetes and insulititis in rats, and increased the total numbers of leukocytes in the peripheral blood and spleen (87). Carboxymethylated curdlan (DB=O) or lentinan increased endotoxin clearance in a dosedependent manner in rabbits (88). PHARMACOKlNFTlCSAND TOXlClrL
,.
- The pharmacokineticsof R-glucans is not well understood, although some data are available. Aminated glucan was derivatized with tyraminyl cellobiose using cyanuric chloride, and radio-iodinated to allow pharmacokinetics studies in mice (89). Smaller molecular weight glucan was found in the kidney and urine, being eliminated more rapidly due to glomerular filtration, while larger glucan was retained primarily within the liver. Tritium labeled scleroglucan, & (DB=1/3) localized to the liver, spleen, kidney and tumor masses in a mouse model (90). When injected into one of a pair of double-grafted tumors, the glucan remained tumor localized but had anti-tumor effects on the distal tumor, presumably via modulation of immune system. Scleroglucan was also found in the blood, liver, and spleen when administered to normal mice, with significant amounts remaining in the liver and spleen for up to four weeks (91). Multiple doses of glucan increased the clearance rate in this system. Radiolabeled Volvariella, 2, glucan (DB=1/6) persisted in the blood (50% of maximum levels still present at 25 hrs), and appeared subject to metabolic conversion in mice to an ionic form which still contained anti-tumor activity (92). The pharmacokinetics of the clinically used anti-cancer adjuvant lentinan (see below) was investigated in normal volunteers and cancer patients (93). Weekly injections of 2 mg lentinan lead to elevated trough levels which might correlate with anti-tumor effects. Blood levels reached a maximum of 50-80 ng/ml at the end of IV infusion (1 mg over 2 hrs), and decreased gradually thereafter (94). Grifolan, 6, (DB=l/3) administered to mice also accumulated at tumor sites, and in various organs, including the spleen (95). Two phases of elimination (Ti12 = 0.5 to 1 hr, and 7 to 12 hrs) were observed following I.V. administration of PGG-glucan to normal volunteers (96), and see below. Joxicitv of Glucans - In general, soluble glucan preparations appear less toxic than particulate glucans. Soluble glucan phosphate derived from S. cerevisiae showed little acute or chronic animal toxicity in preclinical animal evaluations (46,97). A yeast derived glucan was associated with transient hepatomegaly and associated histologic changes, when tested in mice at 250 mgkg I.V. (98). In contrast, systemic administration of microparticulate glucan can cause granuloma formation, hepatosplenomegaly, microembolism, and sensitization to endotoxin (46). Toxicity of a single injection of microparticulate glucan correlated with chronic murine hepatitis infection (99), with minimal effects on virus free mice, indicating that the immune system state my affect responses to glucans. Many of the other in vivo studies referenced above also reported little toxicity for soluble glucan preparations. HUMAN PRFCLlNlCAl ANn CI INlCAL DATA Although available for only small numbers of patients, data on the clinical efficacy of specific I3-1,3 glucans, when used in trauma and surgery patients who are at risk for microbial infection, and as a combination therapy for the treatment of cancer, are available.
-
PGG-Glucaq Clinical development of a genetically modified Saccharomyces cerevisiae glucan, PGG-glucan (Betafectin), is in progress (see below). The precise structure of the material prepared from cells is not known, but certain structural
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features were described. The altered R-1,3 glucan, 14,had DB-1n.4 (100) to DB=1/2 (101), and the average length of the side chains, which consisted of more than one glucosyl unit connected by R-1,6 OH linkages, were 1.1 (100) to 3 (101). PGG-Glucan was proposed to act via interaction with R-glucan receptors 19 where R HO HO on monocytes and neutrophils, stimulating production of cytokines, inflammatory, and colony stimulating OH OH n i factors (101). This conclusion n = 1-3.4; nl 5 1 - 3 correlated with the effects of other RR 1,3 glucans (see above). Most recently (96), the PGG-glucan used in clinical trails was described as a triple helical, branched 0-1,3 glucan (no branching frequency given), possessing branches with 0-1,3 n Immune system linkages, priming, as opposed to direct activation of immune system function and cytokine induction is now considered to be the mechanism OH HO of action of PGG-glucan (96,102). Administration to mice stimulated increased resistance to bacterial challenge in early studies (103). Q PGG-Glucan also enhanced n andwhn2 ereRonot i@] given peripheral-blood microbicidal activity monocytes of human and OH neutrophils (104), nitric oxide production from rat neutrophils (105), HO OH and stimulated hematopoesis alone (106) and in combination with GMCSF (107). PGG-Glucan mode of action presumably involves binding to a 8-glucan receptor (see above section). * . Stimylation of Resistance to w i a l Infection In Patients at R isk - Two recent double blind, placebo-controlled, randomized phase 2 studies examined the safety and efficacy of PGG-glucan in preventing postoperative infection in patients undergoing major thoracic or abdominal surgery. In the first trial results for 17 patients receiving multiple doses (0.5 mgkg) of PGG-glucan were compared to placebo administration (96). Infection incidence and severity was lower in PGG-glucan treated patient group, as was the incidence of postoperative antibiotic usage, and duration of hospital stay including days in intensive care. In a second study, 51 patients in the treatment group were divided into three groups receiving 0.1, 0.5 1.O or 2.0 mg/kg doses (102). Trends toward reduced infection incidence were found with increasing dosage, and data were further analyzed by pooling data for specific groups (see original article for methods of analysis). Two other independent studies reported efficacy of yeast glucan administration to trauma patients. Yeast derived glucan (30 mg I.V. every 12 to 24 hours) decreased the incidence of pneumonia and sepsis in a group of 21 patients treated in a randomized, double blinded, controlled trial (108). The incidence of antibiotic usage was also decreased, as was mortality due to infection and hospitalization stay, including days in intensive care. Another prospective, randomized, double blinded study examined the effects of yeast derived glucan administration to 21 trauma patients given 50 mg /square meter IV phosphate, daily for seven days (109). Septic morbidity was reduced in the treatment group, as was the mortality rate, and slight increases in IL2, but not TNF levels were transiently observed.
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n of Anti-Tumor Re- Lentinan is a purified polysaccharide of 500 kDa extracted from Lentinus edodes, composed of a linear 0-13 glucan with 0-1,6linked branches (DB=l/l2.5), with evidence of branches of greater that one glucosyl unit, and some internal 0-1,6 linkages within the main chain (110). Positive responses were observed when lentinan was used in conjunction with a chemotherapeutic agent for breast ( l l l ) , colo-rectal ( l l l ) , gastric ( l l O , l l l ) , prostate (112) cancers, and malignant ascites and pleural effusions produced from end-stage anal, esophageal, gastric, and pancreatic cancers (113). No positive effects were observed when lentinan was combined with 5FU in the treatment of hepatocellular carcinoma (114). Administration of lentinan stimulates macrophages to produce increased amounts of colony stimulating factors, IL-I, IL-Ill, and other serum factors which modulate activity of the immune system. Sizofiran, 6, (DB=1/3) is a purified polysaccharide of 450 kDa isolated from Schizophyllum. Sizofiran was used in conjunction with radiotherapy for stage I1and stage 111 cervical cancer (1 15-117). The response rate was greatest for stage II cancer (116,117), with no significant difference observed for stage I l l cancer.
Our knowledge of the biology and chemistry of 0-glucans has progressed steadily towards an understanding of 1) glucan structure vs activity, 2) mechanisms of glucan interaction with the immune system and in vivo efficacy, and 3) potential for clinical use. However, even though basic structural properties of natural product glucans are known, they are not structurally defined in the context of the precise structural features interacting with the immune system receptor(s), and the receptor(s) to which glucans bind have not be rigorously defined. The identification of CR3 and other receptor polypeptides for glucans in mammalian systems should ultimately allow a detailed study of glucan structure vs its ability to interact with the immune system, and formulation of an explicit frame work to guide future studies.
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m,
Blologlcal Response Modification by R-D-Glucans
Robert C. Goldman Abbott Laboratories, Pharmaceutical Research Division D-47M/AP9A 100 Abbott Park Rd., Abbott Park, IL 60064-3500
introduction - The mammalian immune defense system is a complex and highly
evolved assemblage, functioning to defend the organism from constant microbial attack from without, and as well disease from within in the form of emerging tumor cells. Although the immune system in its normal state is quite successful in performing its protective role, the slightest decline in functional capacity can predispose the host to infection, and/or lowering of anti-tumor immune surveillance. Furthermore, specific patient populations with normal immune system function are still at risk for infection in certain clinical settings. Thus exogenous stimulation of immune system function is a logical clinical approach to positive intervention. One approach uses endogenous molecules of the immune system (e.g. various cytokines), while another uses exogenous elements, usually of microbial origin. The current report will focus on fungal glucans and their potential for clinical development. b k a r o u d - Zymosan, a cell wall preparation derived from Saccharomyces cerevisiae, has diverse physiological effects, many of which can be beneficial, Cell wall glucan was implicated as the active component in zymosan (l), and subsequent studies revealed that 0-glucans in general did evoke many of the beneficial effects, including stimulation of resistance to microbial infection and activation of anti-tumor responses. Although no glucan-derived products are yet approved for clinical use in the United States, glucans are used elsewhere in specific clinical settings. The structure, biological activity, mode of action, preclinical and clinical data pertaining to glucans will be reviewed.
- Natural product glucans consist of a linear backbone of 0-1,3 linked D-glucopyranosyl groups with varying degrees of branching from the C6 position. Most repolted degrees of branching, i.e., the number of brancheslmain chain residues (DB), should be considered average values with the possibility for heterogeneity in branch distribution as was indicated for glucan isolated from Volvariella (2). Branches are usually only a single glucose residue, although more than one glucose unit may be present in some glucans (3,4), and see PGG-glucan and lentinan below. The repeating unit of natural product glucans with varying branching frequencies include: Alcaligenes (curdlan), Saccharomyces (5), Omphalia OR-OL-~C,Aspergillus (6), Agrocybe (7) and Euglena (paramylon), (DBsO), Omphalia OL-2-111, 1 (DB=1/24), Volvariella, 2 (DBP1/5.5), 1 n 23, 2 n = 5-51a = Fusarium, 3. (DB=1/5), Cryproporus, 9 (DB=1/4), 3s = 3.5* 2v Amanita glucan, 5, (DB=2/7), Grifola (grifolan), L n = l , 8 n=0.5 Omphalia OL-2-1, Schizophyllum (schizophyllan, Schizofiran), and Sclerotinia (scleroglucan), fi (DB=1/3), Omphalia OL-2-11, 2 (DB=1/2), and OL-2, (DB=2/3) glucans. Branching frequency can modulate glucan structure and biological activities (see below). 49
ANNOAL
REPORT[I IN MEDICINAL CHEMI8TRY40
a 9
Copyright 0 l99B by Amdemic Praes. Ino. AU right9 of repmducuon lo any form remmBd.
FJectlonm-Cancer and lnfedloun Diseanes
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Plattner. Ed
The three dimensional structures of lentinan (a), curdlan (9) and 6, schizophyllan, (1 0) were elucidated by X-ray diffraction studies. Triple helical, parallel strands of glucan are hydrogen bonded via the C2 hydroxyls, with higher order structures arising via hydrogen bonding between C4 and C6 hydroxyls. Since the C6 hydroxyls are on the external surface of the triple helix, branches would project away from the helix surface and likely modulate subtle aspects of glucan structure and biological activity. A stereo view of the glucan triple helix is shown (Figure 1). RGlucans can also adopt single chain and single helix forms, as assessed by NMR spectroscopy (11,12). Linear rods, hairpins, circular, globular, and other structures were identified by electron microscopy depending on the glucan sample and mode of preparation (13,14).
Figure 1 . Stereo view of the glucan triple helix.
Early in evolution defense systems evolved to detect and rapidly respond to fungal infection. R-Glucan, a distinguishing component from fungal cells walls, is thus recognized and a defense response initiated. Data are available for specific receptor recognition of glucans in mammalian and non-mammaliansystems, resulting in signal transduction events leading to regulated defense responses.
-
L b n M a ~ n m d h The unit glucan structure binding to a plant glucan receptor was determined, and solubilization and identification by photolabeling of the receptor were reported (15-17). A specific heptasaccharide structure is recognized by the receptor (Kd in the nanomolar range), and elicits plant defense responses. Specific glucan binding proteins also exist in fish and invertebrates, and function to trigger defense systems (18-21). A trimer was the minimal structure interacting with the receptor in salmon, and the intact non-reducing terminus was required (22). The crayfish glucan binding protein may have homology to the glucan binding site of Rglucanase (23).
Mammalian - Complement receptor 3 (CR3)and other receptors are implicated in the
response of mammalian systems to glucans. CR3 is an integrin receptor known to bind several ligands, and apparently 8-glucans as well (24). A role for CR3 in binding glucan
P-D-Olucans
Chap. 14
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is supported by the following: 1) antibody against CR3 blocked responses to glucan (25-27), 2) neutrophils from patients with a deficiency of CR3 were unresponsive to Oglucan (26), and 3) CR3 was isolated from neutrophils by affinity chromatography on Oglucan-Sepharose (26). However, CR3 affinity purified on iC3bSepharose did not bind zymosan and derived glucan in a solid phase assay (28). In addition, stimulation of CR3 with glucan up regulates CR3 (29), and stimulates NK cell cytotoxic activity (24,27,30) presumably via iC3b coated on target cells. Although CR3 plays a role in glucan induced synthesis of monocyte platelet-activating factor (PAF), the actual receptor interacting with glucan was considered to be distinct from CR3 (31). Glucan interaction with CR3 can alter PKC activity, and in the presence of vanadate, can lead to PLA2 activation (33). In addition, mouse peritoneal macrophages have a receptor which reversibly bound aminated O-1,3 glucan, resulting in glucan internalization (32). An anti-idiotype (anti-Id) antibody, prepared using a monoclonal antibody against the B-1,3 glucan laminarin coupled to protein (34), was reported to interact with a O-glucan receptor on monocytes. lodinated anti-Id bound to U937 cells, and reacted with proteins of molecular weights 180 and 160 kDa (35) that consisted of subunits (36) which were differentially regulated (37). A heptasaccharideof undefined structure may represent the unit structure interacting with this receptor (38). N K cell surface proteins which bound yeast wall glucans were also identified in a separate study (39). Corticosteroid treatment of human monocytes increased subsequent phagocytosis of glucan particles as well as glucan stimulated secretion of 0-N-acetylglucosaminidase (40), presumably via modulation of glucan receptors. Retinoic acid and 1,25dihydroxyvitamin D3 also induced 0-1,3 glucan receptors on P388D1 cells (41). In addition, crosslinking of receptors may influence cellular responses to glucans (42). JIFRIVATIVFS
0F GLUCANS
..
o w d G l u u - Glucan physiochemical properties and biological activity can be modulated by chemical modification. Insoluble yeast glucan was converted to water soluble glucan sulfate, 8, (43,44) yielding material with repeating unit empirical formulas of (CgH1005)8-3 S03NH4 and (C6H1005)5*3H2S04. These modified glucans still formed triple helices in solution, and were capable of stimulating bone marrow proliferation and resistance to = Or microbial challenge in mouse models. Soluble, triple helical glucan phosphate with formula B (C6H1005)7.P03H2,10, was also prepared -?' 9 (45,46). Although the extent of substitutions along 0 the 0-1,3 backbone was determined, no experimental data were given as to the precise -p-o - 1Q positions of the sulfate or phosphate groups. Both 0modified glucans contained few if any branches (5). No significant toxicity was observed with glucan phosphate, which is active in promoting wound healing, stimulating resistance to microbial infection, hematopoesis, anti-tumor responses and protection from irradiation (46). O-1,3-Glucans with varying degrees of branching (DB=O, 1i3 and 1/2 see above) were sulfated by reaction with chlorosulfonic acid-pyridine and converted to the sodium salt for analysis of bioactivity (47). Degree of substitution altered the effects of glucans on plasma clotting, complement activation, macrophage stimulation, and mitogenic activity. In addition, the biological effects of a branched glucan were altered by substitution with carboxymethyl groups, suggesting that receptors recognized specific structural features of native glucan (48).
*
[ R:$7] e
Glucan-Drua C o n i u u - Glucan conjugation to cytotoxic drugs modulated toxicity while maintaining the beneficial effects of glucans. Carboxymethylated yeast O-1,3 glucan was coupled to 143-D-arabinofuranosylcytosine(araC) (49) by activating the polysaccharide carboxyl group with isobutylchloroformate and formation of a peptide
Bedion m-cancer e.nd Iniermous Dlseasea
Plattner, Ed.
bond via the 4-NH2 group of araC yielding 11. This conjugate possessed enhanced anti-leukemic activity, and maintained the immunomodulatory activity of the glucan. Mitomycin C was also conjugated to carboxymethylated, linear O-1,3 glucan from Alcaligenes in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide(50) yielding 12. The conjugate was active against P388 leukemia, and sarcoma 180 solid tumors in mice. Mitomycin C conjugated 0 HO to carboxymethylated schizophyllan (DB=1/3) was similarly prepared (51). Carboxymethylation was 1? extensive (degree of substitution of 0.87), occurring predominantly at the C4 or C6 hydroxyls. The conjugate was active against sarcoma 180 Nsolid tumors in mice, and 0 caused less reduction of circulating leukocyte numbers compared to mitomycin C alone.
LN VlTROAND IN VlVO RFSPONSFS TO GI UCANS O-glucans affect immune system functions in vitro including NK cell cytotoxicity (24,27,30,39), CR3 receptor up-regulation (29), production of cytokines (52-57), PLA2 (33), PGE2 (56), arachidonic acid (58),and wound healing factors (57). The relationship between the induction of these and other factors can influence immune system response to bacterial challenge (59). ln vivo effects of glucans include:
.
.
ulation of Resistance to Microbial Challenae - High molecular weight triple helical scleroglucan, 6, increased resistance of mice to challenge with Staphylococcus aureus, Candida albicans and mouse hepatitis virus, with little toxicity (60). Glucan phosphate, lQ,was also active in several animal models (46). The N,Ndimethy laminoethylderivative of paramylon (DB=O) stimulated resistance of mice to challenge with R = H or various microorganisms, whereas carboxymethyl paramylon and paramylon sulfate were less I\C--N& effective (61). Yeast derived 0-1,3-glucan also @ ] . :R acted synergistically with antibiotics via enhanced IL-1 secretion by macrophages, and enhanced OR bone marrow proliferation (62). Soluble, aminated &1,3 glucan, J& modulated cytokine production and protected mice against otherwise lethal bacterial infections (63). Although timing of glucan administration was critical in most studies (also see below for PGG-glucan) long term protection against bacterial challenge was induced in rats (64).
[
ion of HematoDoesk - Glucans also enhanced hematopoesis in animal models, possibly via induction of colony stimulating factors. Scleroglucan, & enhanced bone marrow proliferation when administered to mice (60). Soluble glucan alone, and in combination with human granulocyte colony-stimulating factor enhanced survival and hematopoesis in irradiated mice (65,66). Glucan also abrogated the suppressive effects of pefloxacin on hematopoesis following irradiation (67). Soluble yeast-derived glucan also stimulated hematopoesis with increases in serum levels of granulocyte-macrophage colony stimulating activity (68). Chemically modified glucans (glucan sulfate, g, and glucan phosphate 1p) also stimulated murine bone marrow proliferation following intravenous administration (43,46) in mice.
Chap 14
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on of Anti-Tumor ResDo[lSBS - When examined, the anti-tumor activity of glucans appears due to immune system stimulation as opposed to direct activity against tumor cells. Induction of cytokines, and possible activation of cytotoxic cells, are involved (see below). Scleroglucan, 5, (DB=1/3) increased survival of mice challenged with syngeneic lymphoma, melanoma or adenocarcinoma (60), and inhibited pulmonary metastasis in mice when administered orally or intraperitoneally (69). Glucan also reduced mitomycin C toxicity and promoted an anti-tumor response in a mouse model (51). Branched O-glucans of MW 10,000 and 20,000, were active against Sarcoma 180 in mice, while a related glucan of MW 6000 was inactive, suggesting molecular weight may influence glucan activity in specific systems (70). indicated that tumor Investigation of the mode of action of aminated glucan, regression was stimulated via vascular damage to tumor blood flow, and necrosis via functional T cells and TNF (71). A series of branched glucans isolated from Omphalia (DB=2/3) alone or in were tested for anti-tumor activity in the mouse. OL-2, combination with 5-fluorouracil had activity against solid tumor and ascites forms (72). In addition, derivatives of OL-2 with lesser degrees of branching (1,4, 2) showed potent anti-tumor activity against the solid form of sarcoma 180 mice (73). Another branched glucan isolated from Arnanita, & (DBs217) exhibited significant anti-tumor activity against Sarcoma 180 in mice alone and in combination with mitomycin C (3). Carboxymethylation of the linear glucan prepared from Agrocybe yielded a product with activity against the solid form of Sarcoma 180 in mice (7).
u, a,
of C v w - Cytokine induction by glucans in in vivo animal models was reported in several studies. Administration of OL-2, 6, to mice induced expression of cytokines, including several interleukins, colony stimulating factors, and IL-1 receptor antagonist (IL-1ra) mRNA's (74,75). Schizophyllan, 6, induced IL-1 alpha mRNA comparable to that by OL-2, a, but a weaker level of IL-1 beta mRNA, and did not induce IL-1ra (75). Glucan also acted synergistically with antibiotics via enhanced IL-1 secretion by macrophages, and enhanced bone marrow proliferation (62). Scleroglucan, 5, (DB=1/3) enhanced IL-1 production (76), soluble aminated glucan modulated cytokine production and enhanced IL-1 and TNF levels (63), and paramylon (DB=O) induced IL-1 and IL-6 (77), when administered to mice. O - Glucans also ~ enhanced wound healing via modulation of W of the immune system (46,57,78). The strength of healing surgical incisions was increased by I.V. or topical administration of glucan in a rat surgical model, apparently via enhanced macrophage function and increased cross-linking of collagen (57). Glucan also increased the wound healing rate in a mouse second degree burn recovery model, presumably via alteration of macrophage function (78).
.. Admlnlstration
- Several studies have demonstrated biological activity of glucans administered orally to animals. Orally administered glucan, extracted from Candida albicans (Glucanil, Gluimmun), increased resistance to challenge with Candida albicans or Staphylococcus aureus in a mouse model (79). Glucan increased polymorphonuclear leukocytes and peripheral monocytes levels, and the in vitro candicidal activity of recovered alveolar macrophages and spleen cells. Scleroglucan, 6, (DB-113) increased the number of alveolar macrophages, enhanced their lysosomal enzyme activity, phagocytic activity, and IL-1 production, presumably via effects on Peyer's patch cells (80). The proliferative response of Peyer's patch cells to B and T-cell mitogens was also enhanced (81). Scleroglucan, 5, (DB=1/3) inhibited pulmonary metastasis in mice when administered orally or intraperitoneally (69). ies of Glucan via Oral
.
.
- Glucan in combination with zinc and pig Affects in Other Rtalpaiimmunoglobulin reduced the number of migrating ascarid larvae in the lungs of pigs (82). Glucan isolated from S. cerevisiae suppressed lesions caused by Leishmania in
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and Infectlou~Dieeaaes
PLettner, Ed
mice (83), and acted as an adjuvant during immunization with Leishmania (84) and Trypanosoma (85). Semisoluble I31,3-carboxymethylglucan increased survival in a mouse model of acute massive hemorrhage (86). Branched glucan decreased the cumulative incidence of diabetes and insulititis in rats, and increased the total numbers of leukocytes in the peripheral blood and spleen (87). Carboxymethylated curdlan (DB=O) or lentinan increased endotoxin clearance in a dosedependent manner in rabbits (88). PHARMACOKlNFTlCSAND TOXlClrL
,.
- The pharmacokineticsof R-glucans is not well understood, although some data are available. Aminated glucan was derivatized with tyraminyl cellobiose using cyanuric chloride, and radio-iodinated to allow pharmacokinetics studies in mice (89). Smaller molecular weight glucan was found in the kidney and urine, being eliminated more rapidly due to glomerular filtration, while larger glucan was retained primarily within the liver. Tritium labeled scleroglucan, & (DB=1/3) localized to the liver, spleen, kidney and tumor masses in a mouse model (90). When injected into one of a pair of double-grafted tumors, the glucan remained tumor localized but had anti-tumor effects on the distal tumor, presumably via modulation of immune system. Scleroglucan was also found in the blood, liver, and spleen when administered to normal mice, with significant amounts remaining in the liver and spleen for up to four weeks (91). Multiple doses of glucan increased the clearance rate in this system. Radiolabeled Volvariella, 2, glucan (DB=1/6) persisted in the blood (50% of maximum levels still present at 25 hrs), and appeared subject to metabolic conversion in mice to an ionic form which still contained anti-tumor activity (92). The pharmacokinetics of the clinically used anti-cancer adjuvant lentinan (see below) was investigated in normal volunteers and cancer patients (93). Weekly injections of 2 mg lentinan lead to elevated trough levels which might correlate with anti-tumor effects. Blood levels reached a maximum of 50-80 ng/ml at the end of IV infusion (1 mg over 2 hrs), and decreased gradually thereafter (94). Grifolan, 6, (DB=l/3) administered to mice also accumulated at tumor sites, and in various organs, including the spleen (95). Two phases of elimination (Ti12 = 0.5 to 1 hr, and 7 to 12 hrs) were observed following I.V. administration of PGG-glucan to normal volunteers (96), and see below. Joxicitv of Glucans - In general, soluble glucan preparations appear less toxic than particulate glucans. Soluble glucan phosphate derived from S. cerevisiae showed little acute or chronic animal toxicity in preclinical animal evaluations (46,97). A yeast derived glucan was associated with transient hepatomegaly and associated histologic changes, when tested in mice at 250 mgkg I.V. (98). In contrast, systemic administration of microparticulate glucan can cause granuloma formation, hepatosplenomegaly, microembolism, and sensitization to endotoxin (46). Toxicity of a single injection of microparticulate glucan correlated with chronic murine hepatitis infection (99), with minimal effects on virus free mice, indicating that the immune system state my affect responses to glucans. Many of the other in vivo studies referenced above also reported little toxicity for soluble glucan preparations. HUMAN PRFCLlNlCAl ANn CI INlCAL DATA Although available for only small numbers of patients, data on the clinical efficacy of specific I3-1,3 glucans, when used in trauma and surgery patients who are at risk for microbial infection, and as a combination therapy for the treatment of cancer, are available.
-
PGG-Glucaq Clinical development of a genetically modified Saccharomyces cerevisiae glucan, PGG-glucan (Betafectin), is in progress (see below). The precise structure of the material prepared from cells is not known, but certain structural
Chap 14
Goldman
p-D-OlUcrtna
qOb]
features were described. The altered R-1,3 glucan, 14,had DB-1n.4 (100) to DB=1/2 (101), and the average length of the side chains, which consisted of more than one glucosyl unit connected by R-1,6 OH linkages, were 1.1 (100) to 3 (101). PGG-Glucan was proposed to act via interaction with R-glucan receptors 19 where R HO HO on monocytes and neutrophils, stimulating production of cytokines, inflammatory, and colony stimulating OH OH n i factors (101). This conclusion n = 1-3.4; nl 5 1 - 3 correlated with the effects of other RR 1,3 glucans (see above). Most recently (96), the PGG-glucan used in clinical trails was described as a triple helical, branched 0-1,3 glucan (no branching frequency given), possessing branches with 0-1,3 n Immune system linkages, priming, as opposed to direct activation of immune system function and cytokine induction is now considered to be the mechanism OH HO of action of PGG-glucan (96,102). Administration to mice stimulated increased resistance to bacterial challenge in early studies (103). Q PGG-Glucan also enhanced n andwhn2 ereRonot i@] given peripheral-blood microbicidal activity monocytes of human and OH neutrophils (104), nitric oxide production from rat neutrophils (105), HO OH and stimulated hematopoesis alone (106) and in combination with GMCSF (107). PGG-Glucan mode of action presumably involves binding to a 8-glucan receptor (see above section). * . Stimylation of Resistance to w i a l Infection In Patients at R isk - Two recent double blind, placebo-controlled, randomized phase 2 studies examined the safety and efficacy of PGG-glucan in preventing postoperative infection in patients undergoing major thoracic or abdominal surgery. In the first trial results for 17 patients receiving multiple doses (0.5 mgkg) of PGG-glucan were compared to placebo administration (96). Infection incidence and severity was lower in PGG-glucan treated patient group, as was the incidence of postoperative antibiotic usage, and duration of hospital stay including days in intensive care. In a second study, 51 patients in the treatment group were divided into three groups receiving 0.1, 0.5 1.O or 2.0 mg/kg doses (102). Trends toward reduced infection incidence were found with increasing dosage, and data were further analyzed by pooling data for specific groups (see original article for methods of analysis). Two other independent studies reported efficacy of yeast glucan administration to trauma patients. Yeast derived glucan (30 mg I.V. every 12 to 24 hours) decreased the incidence of pneumonia and sepsis in a group of 21 patients treated in a randomized, double blinded, controlled trial (108). The incidence of antibiotic usage was also decreased, as was mortality due to infection and hospitalization stay, including days in intensive care. Another prospective, randomized, double blinded study examined the effects of yeast derived glucan administration to 21 trauma patients given 50 mg /square meter IV phosphate, daily for seven days (109). Septic morbidity was reduced in the treatment group, as was the mortality rate, and slight increases in IL2, but not TNF levels were transiently observed.
ROkpl P
u.
'./
xi
a,
rn
Phttner, Ed
section m-cancer and InfeaLious D l s e a s ~ ~
n of Anti-Tumor Re- Lentinan is a purified polysaccharide of 500 kDa extracted from Lentinus edodes, composed of a linear 0-13 glucan with 0-1,6linked branches (DB=l/l2.5), with evidence of branches of greater that one glucosyl unit, and some internal 0-1,6 linkages within the main chain (110). Positive responses were observed when lentinan was used in conjunction with a chemotherapeutic agent for breast ( l l l ) , colo-rectal ( l l l ) , gastric ( l l O , l l l ) , prostate (112) cancers, and malignant ascites and pleural effusions produced from end-stage anal, esophageal, gastric, and pancreatic cancers (113). No positive effects were observed when lentinan was combined with 5FU in the treatment of hepatocellular carcinoma (114). Administration of lentinan stimulates macrophages to produce increased amounts of colony stimulating factors, IL-I, IL-Ill, and other serum factors which modulate activity of the immune system. Sizofiran, 6, (DB=1/3) is a purified polysaccharide of 450 kDa isolated from Schizophyllum. Sizofiran was used in conjunction with radiotherapy for stage I1and stage 111 cervical cancer (1 15-117). The response rate was greatest for stage II cancer (116,117), with no significant difference observed for stage I l l cancer.
Our knowledge of the biology and chemistry of 0-glucans has progressed steadily towards an understanding of 1) glucan structure vs activity, 2) mechanisms of glucan interaction with the immune system and in vivo efficacy, and 3) potential for clinical use. However, even though basic structural properties of natural product glucans are known, they are not structurally defined in the context of the precise structural features interacting with the immune system receptor(s), and the receptor(s) to which glucans bind have not be rigorously defined. The identification of CR3 and other receptor polypeptides for glucans in mammalian systems should ultimately allow a detailed study of glucan structure vs its ability to interact with the immune system, and formulation of an explicit frame work to guide future studies.
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