γ-Propoxy-sulfo-lichenin, an antitumor polysaccharide derived from lichenin

PHARMACEUTICA i\CTAHELVETIAE ELSEVIER

Pharmaceutics

Acta Helvetiae

y-Propoxy-sulfo-lichenin,

70 (1995) 25-31

an antitumor polysaccharide from lichenin ’

derived

Andreas Hensel * Taunusring 16, 63755 Alzenau / (Received

1 December

1993; accepted

Ufr.,Germany 28 August

1994)

Abstract A water-soluble semisynthetic polysaccharide, y-propoxy-sulfa-lichenin (PSL), was prepared by reaction of propansultone with lichenin, a natural occurring P-I.3/1.4-linked glucan originating from Cetraria sp. PST_ represents a class of mixed-linked @-glucans with long and hydrophilic side chains in position C-6 of the glucan backbone. PSI_ with a degree of substitution of 0.8 and an average molecular weight of 250 kDa exhibited a strong antitumor activity in doses of 25 and 5 mg/kg against solid sarcoma 180 (100% resp. 82% tumor inhibition). The antitumor activity of PSL was shown to be dependent on the dimension of the molecule: the higher the average molecular weight, the higher was the inhibition rate obtained in the antitumor assay. No antitumor effect was observed by using a pretreatment of animals prior to transplantation of sarcoma 180. On syngenic DBA/2-MC.SCl fibrosarcoma PSI_ inhibited tumor growth by about 88% at a concentration of 25 mg/kg. PSI_ failed to exhibit any direct cytotoxic effects on hormone-independent MDA-MB 231 mammacarcinoma. For PSI_ an indirect antitumor effect via modulation of the host mediate immune defence is postulated. Keywords:

Antitumor

polysaccharides;

Biological

response

modifier;

Lichenin;

1. Introduction

Various kinds of biological response modifiers (BRM) have been developed in the last decades for therapy of cancer. Within the large group of polysaccharides tested in this field a huge variety of different structures have been proved to affect the progress of tumor growth in many experimental systems (Whistler et al., 1976). Typical for the activity of these substances is the absence of any direct cytotoxic effects; the pharmacological mechanisms leading to the antitumor efAbbreuations: BRM, biological response modifier; GPC, gel-permeation chromatography; kDa, kilo dalton; MWCO, molecular weight cut off; NMR, nuclear magnetic resonance; PSI_, y-propoxy-sulfolichenin, sodium salt * Corresponding author. Present address: Friedrich-AlexanderUniversity, Pharmaceutical Biology, Staudtstr. 5, D-91508 Erlangen, Germany. ’ Work performed at the University of Regensburg, Faculty of Chemistry and Pharmacy, UniversitZtsstr. 31, 93053 Regensburg, Germany. 0031-6865/95/$09.50 0 1995 Elsevier SSDI 0031-6865(94)00048-4

Science

B.V. All rights resewed

y-Propoxy-sulfa-lichenin;

Sarcoma

180

fects seem to be manyfold for such polysaccharides: stimulating effects on T-lymphocytes (Hamuro et al., 1978; Ajinomoto et al., 19851, activation of macrophages (Seljelid et al., 1981) as well as anticomplementary effects (Yamada et al., 1985; van der Nat et al., 1987) are described. No reports are available dealing with specific antigen-antibody reactions enhanced by these BRM. Within a world-wide screening for such antitumor polysaccharides many active products have been isolated in the last years from higher plants (Kraus et al., 1988), yeasts (Diller et al., 1963) and - the most active ones - from fungi (Chihara et al., 1970; Dais and Perlin, 1982; Misaki et al., 1984; Bruneteau et al., 1988; Gomaa et al., 1992). Concerning the structural features especially /?-1.3-glucans with side chains in position 6 of the backbone showed marked antitumor activity (Tabata et al., 1981; Dais and Perlin, 1982; Bruneteau et al., 1988; Gomma et al., 1992). Because it is often hard to obtain high amounts of such pharmacologically active glucans from biological sources it is worth to search for a cheap and easy way to obtain highly active

A. Hensel/Pharmaceutica

26

Acta Heluetiae 70 (1995) 25-31

antitumor polysaccharides in high yield by means of semisynthetic modifications of commercially available glucans. Within a structure-activity relationship it was shown that substitution of linear P-glucans with long and hydrophilic side chains - which do not need to be of carbohydrate nature - can enhance a basic antitumor activity of the substrate manyfold (Hensel et al., 1988; Hensel and Franz, 1989). During the search for such new, semisynthetic glucans y-propoxy-sulfo-lichenin (PSL) was prepared by using lichenin, a P-1.3/1.4-glucan from Cetraria islandica L. and Cetraria tenuifolia Retz (Parmeliaceae) as substrate. The use of lichenin for preparation of new, semisynthetic derivatives is justified by a basic antitumor activity described in the literature (Helberger et al., 1946; Hensel and Franz, 1989). Nevertheless sulfoalkylated derivatives of lichenin, substituted at C-2 and, preferentially, at C-6 are described to be inactive when tested against Sarcoma 180 tumor (Demleitner et al., 1992a). The aim of the present study was the development of a structure-activity relationship to test a similar, but to a lower degree substituted propoxy-sulfonated lichenin with side chains exclusively at C-6 against several experimental tumor systems. Because of the positive results obtained from these experiments various investigations on the structural features, e.g. the existence of helical structures, dependence of activity from the chain length etc. were carried out and described in this paper.

2. Experimental procedures 2.1. Materials Lichenin (Batch No. A6768561 was obtained from Carl Roth GmbH + CoKG (Karlsruhe, Germany) and propansultone was available from Jansen (Belgium). All other chemicals and reagents used in this paper were purchased from Merck (Darmstadt, Germany). Materials for chromatography (standard dextrans, Superose TM-6 and Sephacryl-S-400) were obtained from Pharmacia (Germany). CDl, BDFl and DBA/2 mice, 7 to 9 weeks old, were purchased from Charles River Wiga (Sulzfeld, Germany). Animals were kept under standard conditions and received standard diet (Altromin) and tap water ad libitum. 2.2. General procedures IR data were spectrophotometer.

obtained

with a Beckman

Acculab

3

Elemental analysis (C, H) was accomplished by the microlaboratory of the University of Regensburg and the S-determination by University of Stuttgart, both Germany. 13C-NMR spectra (Laboratoire de Resonance Magnetique Nucleaire de Universite de Montreal, Montreal, Canada) were obtained using lo-mm-diameter tubes with 40 mg PSL dissolved in 3 ml D,O. Data were collected under conditions of proton decoupling, using a Bruker spectrometer (model WH 400) operated in Fourier-transform mode with 730 pulse angle at 100.62 MHz with about 10000 scans at 353 Kelvin. The reference for the chemical shift values was external Dioxane at 67.4 ppm: carbon atoms of 1-6linked glucose residues (Dais and Perlin, 1982): C-l 103.5, C-2 74.6, C-3 76.1, C-4 79.9, C-5 75.5, C-6 61.5 ppm; carbon atoms of 1-3-linked glucose-residues: C-l 103.7, C-2 74.1, C-3 85.7, C-4 69.4, C-5 76.8, C-6 62.1 ppm; alcoxylated carbon: C-6 70.1; substituents: C-l 49.3, C-2 25.6, C-3 71.2 ppm. 2.3. Preparation of PSL Preparation was performed according to Helberger et al. (1946) by using 1.0 g lichenin as substrate and 10 g propansultone as reactant. After the finished reaction the resulting raw product was dialysed against flowing tap-water for 24 h (MWCO: 3.5 kDa) and following an excessive dialysation for at least 3 days against flowing purified water was performed. The dialysate was centrifuged (1500 X g for 10 min) to get rid of undissolved material, and the resulting slightly opalescent supernatant was concentrated under vacua at a temperature not exceeding 40°C. The pH of the solution was adjusted to 7.5 (kO.05) with NaOH 0.1 mol/l and the product was finally freeze dried. Molecular formula: C,H,,.,O,S,,Na,., for PSL with DS 0.8; theor.: C: 36.6%, H: 5.2%, S: 8.7%; found: C: 36.0%, H: 5.1%, S: 8.7%; 3400/ 2900/ 2870/ 1720/ 1680/ 1380/ 1200/ IR: 1050/ 895/ 795/ 740 cm-‘. 2.4. Chroma tograpic conditions Calibration of chromatograpic systems used were performed by using standard dextrans with average molecular weights of 500, 250, 200, 70 and 40 kDa as well as dextran blue for determination of the void volume.

A. Hen.sel/ Pharmaceutics Acta

Determination of the average molecular weight of PSL and analysis on multiple aggregation was carried out on a Superose TM-6 column (1 X 30 cm> at a flow rate of 30 ml/h. Injection volume was 100 ~1 of a 1% (w/v) solution in the respective eluent. The colum was eluted with NaCl 0.1 mol/l or, for aggregation analysis, with urea 8 mol/l. Fractions of 0.5 ml were collected by using a Frac 100 (Pharmacia) and checked on carbohydrate content by the antrone method (Morris, 1948). 2.5. Preparation of PSL derivatives with various chain length 100 mg PSL were disolved in 4 ml water and autoclaved for 20 min at 121°C. In several chromatograpic runs 1 ml of the hydrolysate was subjected to a calibrated Sephacryl S-400 semi-preperative column (87 x 1.6 cm> and eluted with NaCl 0.1 mol/l at a flow rate of 20 ml/h. Aliquots (0.1 ml) of the fractions (2.0 ml> were checked on carbohydrate content by the antrone method (Morris, 1948). Fractions containing PSL derivatives in the molecular weight range of 1000 to 250 kDa, 250 to 25 kDa and 25 to 6 kDa were pooled, dialyzed against purified water (MWCO: 3.5 kDa) and freeze dried.

Helvetiae70 (1995) 25-31

27

Animals were sacrificed at day 30 after tumor inoculation and the tumors excised and weighed. Antitumor activity against DBA/2-MC.SC-1 fibrosarcoma was performed according to Gomma (1992). This tumor had been induced by S.C.application of 3-methylcholanthren (0.5 mg per mouse) in female DBA/2 mice. The tumor was maintained by S.C.transplantation of tumor pieces 8 about 1 mm3 every 4 to 5 weeks in female DBA/2 mice. The same procedure was applied for routine testing of PSL, using 10 animals per dose group. The inhibition rate was calculated as follows: (C - T)/C

x 100.

T: average tumor weight of treated group; C: average tumor weight of control group. The regression rate was determined as the ratio of the number of tumor-free animals to the total number of treated animals. Determination of significant results was performed by using the Student’s f-test (double-sided) at a P < 0.05 level of significance. In-vitro assay on direct cytocidal potential of PSL was carried out according Cailleau et al. (1974) and Lippman et al. (1977).

2,6. Assay of antitumor activity: biological methods Antitumor activity against sarcoma 180 was performed as described by Gomaa et al. (1992). Sarcoma 180 was a gift of Dr. Bogden (Mason Research Institute, Worchester, MA, USA). Sarcoma 180 was maintained by using intraperitoneal passages of ascites fluid (5 x lo6 tumor cells) in female BDFl mice every seven days. Screening on antitumor activity of polysaccharides was performed by subcutanous inoculation of 0.1 ml ascites (5 x lo6 tumor cells) into the groin of female CD1 mice. This inoculation was counted to be day zero of the experiment. One group of mice existed of 10 animals. Standard treatment was the daily application of PSL from day 1 to 10, starting 24 h after tumor inoculation. From day 11 to day 30, which means up to the end of the experiment, no more application was performed. Changes to this standard cycle were performed for testing the effect of a different administration cycles as described under results (application three times a week, starting at day 1 and continuing until day 30; unit dose application at day 1 with no subsequent treatment; pretreatment 10 days prior tumor inoculation; treatment only at days 1 to 3, or 4 to 6, or 7 to 10). For evaluation of tumor growth the tumor area (length X width) was measured with a caliper every 10 days.

3. Results 3.1. Preparation and characterization of PSL

For preparation of y-propoxy-sulfo-substituted derivatives of lichenin, the substrate was reacted in strongly alkaline medium with propansultone (Fig. 1). Extensive dialysis of the product was performed to avoid any contamination with residual reactants or low molecular side products caused by degradation of the substrate. Additional purity control by using infrared spectroscopy did not show any signs of the typical bands of esters of sulfonic or sulfuric acid nor these typical for alkylated sulfuric acids. 13C-NMR indicated the presence of an exclusive substitution of the hydroxyl groups of C-6 of glucose

Fig. 1. Synthesis of PSL from lichenin and propansulton medium.

in alkaline

28

A. Hensel/ Pharmaceutics Acta Helcetiae 70 (1995) 25-31

residues (signal at 70.1 ppm). No signs of substitution at C-2, C-3 or C-4 were detected. In contrast to the unsubstituted lichenin, PSL was shown to be freely soluble in water up to 5 mg/ml. Elemental analysis resulted in a content of about 8% sulfur, corresponding to a degree of substitution of 0.8. Determination of the molecular weight of PSL by using gel permeation chromatography on a dextran-calibrated in a single peak with a SuperoseTM column resulted maximum at 252 kDa. Because some known antitumor polysaccharides tend to appear in specific multiple aggregates, e.g. triple helices, PSL was examined on such non-covalent aggregates. Therefore PSL was treated in aqueous solution with urea 8 mol/l, a strong chaotropic reagent. An aliquot of the PSL/urea-containing solution was subjected to the Superose column and the column was eluted with urea 8 mol/l. The polysaccharide was detected in exactly the same fractions as it was when elution was performed without urea. The existence of multiple helical structures between PSL molecules can therefore be excluded. 3.2. Antitumor effect of PSL against sarcoma 180 For a first screening on antitumor activity, PSL was administered in doses of 25, 5 and 1 mg/ kg against allogenic sarcoma 180 solid-type tumor in CD1 mice using groups of 10 mice per dose group. Using a treatment cycle starting 24 h after S.C. transplantation (at day 0) of tumor cells, PSL was given once a day intraperitonally for consecutive 10 days. No further administration was performed from day 11 to day 30. No detectable toxicity was observed even at high doses. As shown in Fig. 2, a dramatic decrease of the tumor area occurred after day 10 at doses of 25 and 5 mg/kg. Evaluation of the tumor weights after sacrificing of the

-

Ccmtlol

--t

I mgikg

-

5 rmJ/kQ

----t

25 mg/kg

Table 1 Antitumor effect of PSL on Sarcoma 180; PSL (25, 5 and 1 mg/kg) was applied from day 1 to 10; total test time: 30 days Substance

Dose (mg/kg)

Tumor weight

Inhibition (%)

Regression

Significance

100 82 13 _

lO/lO

< 0.01 < 0.01 n-s. _

(g) PSL PSL PSL Control

25 5 1 -

0.00 0.77 3.64 4.18

8/9 4/10 o/10

animals at day 30 gave evidence (Table 1) of 100% inhibition of tumors and complete regression in all animals in the 25-mg group. A minor effect with 82% inhibition and a high rate of regression was observed at 5 mg/kg. However, PSL showed only little effect at doses of 1 mg/ kg. 3.3. Antitumor effect against methylcholantren-induced fibrosarcoma In order to get information on the antitumor activity of PSL against syngenic tumor systems which are known to be less sensitive and therefore hard to influence by antitumor therapy, doses of 25 and 5 mg/ kg PSL were administered against methylcholantren-induced fibrosarcoma of DBA/2 mice. Table 2 shows higly significant effects in both doses with greater activity at 25 mg/kg: complete tumor regression was observed in two from nine animals; the inhibition rate was calculated to be 88%. 3.4. Antitumor effect of PSL with various chain lengths To study the influence of the chain length of PSL on antitumor activity, fractions with different molecular weights were prepared by mild acid hydrolysis of a solution of PSL at pH 5.0 and 121°C for 20 min. This hydrolysate was fractionated on a calibrated semi-preparative Sephacryl-S-400 GPC-column. Three samples with different molecular weight (Table 3) were ob-

Table 2 Antitumor effect (25 and 5 mg/kg)

of PSL on DBA,GMC.SC-1 Fibrosarcoma; PSL was applied from day 1 to 30 three times a week

Substance

Dose (mg/kg)

Tumor weight

PSL PSL Control

25 5 -

0.50 2.61 4.28

Inhibition (%)

Regression

Significance

88 39 -

2/9 o/10 o/13

< 0.0001 < 0.01

(g) Fig. 2. Antitumor effect of PSL in doses of 25, 5 and 1 mg/kg on sarcoma 180 or CD1 mice. Treatment was performed from day 1 to 10.

A. Hensel/ Pharmaceutics Acta Helvetiae 70 (1995) 25-31

29

Table 3 Antitumor effect of PSL on Sarcoma 180; PSL (5 mg/kg) was applied from day 1 to 10; total test time: 30 days Substance

Molecular weight (kDa)

Tumor weight (g)

Inhibition (%I

Regression

Significance

PSL PSL PSL Control

1000-250 250-25 25-6 _

0.00 1.38 1.46 4.18

100 67 65

lO/lO 6j9 5/7 o/15

< 0.01 < 0.02 < 0.05 _

tamed by collecting different fractions. The results of these PSL derivatives in the antitumor assay against sarcoma 180 are shown in Table 3. A much higher activity of the highest molecular weight weight fraction (100% inhibition and complete tumor regression in all animals) was observed at 5 mg/ kg doses compared to the medium- resp. low-molecular weight derivatives. Therefore a clear dependence of the antitumor activity of PSL from the moIecuIar weight can be claimed. It seems not surprising that the high molecular fraction has a higher activity compared to the non-fractionated PSL against sarcoma 180 (see also Table 1). 3.5. Antitumor effect of PSL against sarcoma 180 in different treatment cycles

Differing from the standard IO-day treatment cycle (day 1 to 10) used in the sarcoma 180 assay some different application procedures were performed (Table 4). Administration of 5 mg/ kg PSL three times a week over a 30-day test cycle and sacrificing the animals at day 30 with no postadministration period resulted in a 96% inhibition of tumor mass. Application of a unit dose of 50 mg/kg at day 1 was slightly less effective (90% inhibition and a minor regression). To get an idea whether there is a certain time interval in the first 10 days after tumor transplantation Table 4 Antitumor effect of PSL on Sarcoma 180; PSL was applied different treatment cycles; total test time: 30 days Substance

Dose (mg/kg)

Treatment

Tumor weight (g)

Inhibition (%)

Regression

PSL Control

5 -

day l-10 day l-10

0.77 5.66

82 a -

g/9 2/15

PSL

5

0.24

96 b

7/g

PSL

50

day l-30 3 x per week day 1 only

0.73

90 a

4/7

PSL

5

day l-3

3.08

26 =

30

PSL PSL Control

5 5 -

day 4-6 day 7-10 day l-10

2.84 3.37 4.18

32 c 19 c _

2/7 4/7 o/15

a Significant P < 0.001; b significant P < 0.01; ’ not significant.

in

which is essential for an effective PSL application, 5 mg/ kg doses of the drug were applied from day 1 to 3, 4 to 6, or 7 to 10. A marked decrease of antitumor activity compared to the standard application was observed in each of these cases. This may be explained by the reduction of the effective dose to one third. So no special time interval during the first 10 days for application of PSL seems to be preferred to induce a pronounced antitumor effect against sarcoma 180. 3.6. Effect of pretreatment with PSL Because of the effectiveness of pretreatment of animals prior to tumor transplantation, which is described in various cases for antitumor polysaccharides (Sasaki et al., 1978; Misaki et al., 1984), PSL was given in a dose of 5 mg/kg for 10 days to tumor-free CD1 mice. Tumor cells were transplanted 24 h after the last application. No further treatment was carried out for the next 30 days. Evaluation of the tumor weights did not show any positive antitumor effect in comparison to the control group. 3.7. Direct cytotoxic activity of PSL on tumor cells in vitro

To examine PSL on a possible direct cytotoxic effect on tumor cells in-vitro testing was carried out by incubation of 5, 25 and 125 pg/ml PSL together with hormone-independent MDA-MB 231 mammacarcinoma cells. For evaluation of a possible antitumor effect the parameter cell growth inhibition and c3H>thymidine incorporation were measured after a 4-day cultivation period. No significant changes between the control group and PSL-treated groups were observed in any case. This indicates clearly the absence of any direct cytocidal potential of PSL.

4. Discussion In the field of development and screening of nonnatural occurring BRMs, few reports are available on

30

A. Hensel/ Pharmaceutics Acta Helvetiae 70 (1995) 25-31

semisynthetic antitumor polysaccharides (Hamuro et al., 1971; Matsuzaki et al., 1986; Hensel and Franz, 1989; Demleitner et al., 1992a,b). The easy way of preparation, the possibility of radio labeling for pharmacokinetic studies and the possibility for specific changes by drug modeling can make such substances to a potent tool for research on BRM. In the present study PSL, a semisynthetic licheninbased polysaccharide was synthesized, characterized and tested for antitumor activity. In contrast to the substrate lichenin, which is nearly insoluble in water at room temperature, PSL is freely soluble up to 5 mg/ ml in cold water due to the salt form of the molecule and the strongly hydrophilic side chains. Reasonable water solubility is one of the important properties potential antitumor polysaccharides must possess in order to guarantee an unproblematic application as solution for injection. Only one case of an antitumor polysaccharide is described where a - water-insoluble - glucan was administered orally with marked antitumor effects (Suzugi et al., 1990); the more conventional way of treatment is injection of an aqueous solution due to the minimal resorption of these macromolecules by oral application. It is interesting that the way of preparation of PSL leads to a product substituted only at position 6 of the glucan backbone. Therefore PSL seems to be different compared with products synthesized by Demleitner et al. (1992b) with an additional substitution at C-2, a higher degree of substitution and a higher average molecular weight. It is interesting that no multiple aggregates of basic PSL molecules have been detected. The existence of helicoid structures is described to be essential for antitumor effects of certain cy-glucans, such as lentinan (Bluhm et al., 1977) or pestalotan (Misaki et al., 1984). However, this finding on PSL, as well as results on glucans from Phytophthoru species (Kraus et al., 1992) and Lentinan (Maeda et al., 1988) indicate clearly that such supermolecular organisation of the macromolecules is not a supposition for a marked antitumor effect. Looking at the efficacy of PSL against sarcoma 180, strong antitumor effects were observed which are dose dependent: the higher the dose, the higher the effect. Also against syngenic methylcholantren-induced fibrosarcoma doses of 2.5 mg/kg were much more effective than lower PSL concentrations. It is speculative if a further increase of the doses would lead to the optimal curves described for other antitumor polysaccharides (Sasaki et al., 1978) and characterized to be a

general difference to the action of direct cytotoxic agents. Because of ethical reasons with animal experiments such tests have not been carried out during this study. A further increase in activity of PSL was observed when PSL fragments with high molecular weight (1000-250 kDa) were used in the bioassay: the enhancement of the molecular dimension was related to an increase in tumor inhibition and regression rates, a phenomenon observed with most antitumor polysaccharides tested up to now (Misaki et al., 1984; Sasaki et al., 1978). This effect may be due to better recognition of the huge molecules by the target cells or the increased time of existence in the body. For glucans which are only effective in the state of helical structures a minimum molecular weight of 100 kDa is described to hold for example a triple helix. Because of the absence of any direct cytotoxic effects of PSL against tumor cells a host mediate action is claimed for this polysaccharide. However, the ineffectiveness of PSL when using pretreatment of animals prior to tumor transplantation does not seem to be consistent with other reports on positive action by such a way of treatment by immunactive polysaccharides (Sasaki et al., 1978; Misaki et al., 1984). Only carboxymethyl-pachymaran is described to be not effective by using pretreatment in spite of a pronounced antitumor effect of this substance when applied after tumor transplantation (Hamuro et al., 1971). It may be worth to discuss a completely different mode of action for such hydrophilic, semisynthetic glucans in contrast to the natural occurring neutral antitumor polysaccharides; this may be due to the changed binding capacities to cell surfaces because of the different polarities of the side chains or to different kinetic parameters of the molecules. Concerning the development of structure-activity relationship it is interesting to note that changes in the substitution pattern of such sulfo-alkylated products as it is observed between PSL and the derivatives prepared by Demleitner et al. (1992b) lead to different efficacy. Products substituted at C-2 and C-6 of the backbone do not show significant efficacy in contrast to PSL, being a potent antitumor polysaccharide against the tested tumor systems. Nevertheless PSL seems to be quite different from other antitumor polysaccharides it seems worth to apply further work in the development of this polysaccharide derivate to obtain more information concerning the mode of action, to get a substance into hands as a possible potent tool in the future treatment of cancer.

A. Hensel/ Pharmaceutics Acta Helvetiae 70 (1995) 25-31

Acknowledgments

The author is thankful for the possibility to perform studies at the laboratories of Prof. Dr. G. Franz, Department of Chemistry and Pharmacy, University of Regensburg. The author is grateful to Prof. Dr. H. SchGnenberger and coworkers for performance of the in-vitro studies.

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