Soluble phosphorylated glucan: methods and compositions for treatment of neoplastic diseases

A new class of soluble phosphorylated glucans is described as well as the process for making the same. According to one embodiment, the soluble phosphorylated glucan is derived from the yeast Saccharomyces cerevisiae. The soluble phosphorylated glucans are useful for prophylactic and therapeutic applications against neoplastic, bacteria, viral, fungal and parasitic diseases. The soluble phosphorylated glucans are used either alone or in combination with a known antimicrobial agent for prophylactic and therapeutic antimicrobial applications. Additionally, they may be administered either alone or as a non-toxic adjuvant, in combination with chemotherapy. The soluble phosphorylated glucans are also useful for stimulating macrophage cells, either in vivo or in vitro, to produce a cytotoxic/cyctostatic factor effective against cancer cells.

TABLE OF CONTENTS 
1. Field of the Invention 
2. Background of the Invention 
2.1. Immunobiological ctivity of Particulate Glucans 
2.2. Adverse Side Effects of Particulate Glucans 
2.3. Unsuccessful Attempts to Solubilize Particulate Glucans 
3. Summary of the Invention 
4. Brief Description of the Figures 
5. Detailed Description of the Invention 
5.1. Process for Preparation of Soluble Phosphorylated Glucan 
5.2. Characterization of Soluble Phosphorylated Glucan 
5.2.1. Elemental Composition 
5.2.2. Structural Configuration 
5 2.2.1. Molecular Sieving 
5.2.2.2. Nuclear Magnetic Resonance Spectroscopy 
5.3. Non-toxicity, Non-pyrogenicity and Non-Immunogenicity of Soluble 
Phosphorylated Glucan 
5.3.1. Non-Toxicity 
5.3.2. Non-Pyrogenicity 
5.3.3. Non-Immunogenicity 
5.4. Uses of Soluble Phosphorylated Glucan 
5.5. Routes and Methods of Administration 
6. Preparation of soluble Phosphorylated Glucans 
6.1. Preparation from Particulate Glucan Obtained from Saccharomyces 
6.2. Preparation from Coriolus versicolor 
6.3. Preparation from Sclerotium 
7. Immunobiological Properties of Soluble Phosphorylated Glucans 
7.1. Modification of Enhanced Susceptibility to OpportunisticInfections in 
Immunosuppressed Animals 
7.1.1. Enhanced Survival 
7.1.2. Enhanced Resistance of Immuno-Suppressed Mice to Escherichia coli 
Infection 
7.2. Enhancement of Macrophage Phagocytic Activity 
7.3. Enhancement of Macrophage Secretory Activity 
7.4. Enhancement of Anti-Tumor Cytotoxin Production by Macrophages In Vitro 
7.5. Enhancement of Kupffer Cell Tumoricidal Activity 
7.6. Enhancement of Proliferation of Bone Marrow Cells In Vitro 
7.7. Enhancement of Splenocyte Response to Mitogens 
7.8. Modification of Metastatic Liver Disease 
7.9. Ability of Soluble Phosphorylated Glucan to Prevent Cyclophosphamide 
Induced Leukopenia 
7.10. Direct Cytostatic Effects on Neoplastic Cells 
7.10.1. Cytostatic Effect on Proliferation of Lymphocytic Leukemia Cells In 
Vitro 
7.10.2. Cytostatic Effect on Proliferation of Sarcoma and Melanoma Cells In 
Vitro 
1. FIELD OF THE INVENTION 
The present invention relates to pharmaceutical compositions and methods 
for therapeutic treatment of neoplastic diseases. More particularly, the 
invention relates to compositions and methods for treatment of a variety 
of neoplastic diseases using a novel class of soluble phosphorylated 
glucans in which the poly-[beta-(1-3)glucopyranose] chains are 
phosphorylated in varying degrees. 
2. BACKGROUND OF THE INVENTION 
The term "glucan" refers generically to a variety of naturally occurring 
homopolysaccharides or polyglucoses, including polymers such as cellulose, 
amylose, glycogen, laminarians, starch, etc. Glucan encompasses branched 
and unbranched chains of glucose units linked by 1-3, 1-4, and 1-6 
glucosidic bonds that may be of either the alpha or beta type. 
As defined herein, "particulate glucan" designates a water-insoluble 
particulate (about 1-3 microns) polyglucose such as that derived from the 
cell wall of the yeast Saccharomyces cerevisiae. Particulate glucan is 
macromolecular and comprises a closed chain of glucopyranose units united 
by a series of beta-1-3 glucosidic linkages. (Hassid et al., 1941, J. 
Amer. Chem. Soc. 63: 295-298; Di Luzio et al., 1979, Int'l J. Cancer 24: 
773-779). X-ray diffraction studies have demonstrated that particulate 
glucans exist in the form of triple-stranded helices. (Sarko et al., 1983, 
Biochem. Soc. Trans. 11: 139-142). 
2 1 IMMUNOBIOLOGICAL ACTIVITY OF TICULATE GLUCANS 
Particulate glucan is a potent activator of the macrophage/monocyte cell 
series, complement, as well as of B cell lymphocytes. Thus, particulate 
glucan has profound effects on both the reticuloendothelial and immune 
systems. 
Previous studies have demonstrated that in vivo administration of 
particuaate glucan to a variety of experimental animals induces a number 
of profound immunobiological responses, including the following: (1) 
enhanced proliferation of monocytes and macrophages (Deimann and Fahimi, 
1979, J. Exper. Med. 149: 883-897; Ashworth et al., 1963, Exper. Molec. 
Pathol., Supp. 1: 83-103); (2) enhanced macrophage phagocytic function 
(Riggi and Di Luzio, 1961, Am. J. Physiol. 200: 297-300); (3) enhanced 
macrophage secretory activity (Barlin et al., 1981, in Heterogeneity of 
Mononuclear Phagocytes, Forster and Landy, eds., Academic Press, New York, 
pp. 243-252); (4) increased macrophage size (Patchen and Lotzova, 1980, 
Exper. Hematol. 8: 409-422); (5) enhanced macrophage adherence and 
chemotactic activity (Niskanen et al., 1978, Cancer Res. 38: 1406-1409); 
and (6) enhanced complement activation (Glovsky et al., 1983, J. 
Reticuloendothel. Soc. 33: 401-413). Increased cytolytic activity against 
tumor cells has been demonstrated in macrophages from animals and man 
treated with particulate glucan both in vivo (Mansell and Di Luzio, 1976, 
in "The Macrophage in Neoplasia", Fink, ed., Academic Press, New York, pp. 
227-243) and in vitro [Schultz et al., in Immune Modulation and Control of 
Neoplasia by Adjuvant Therapy, M.A. Chirigos, ed. Raven Press, New York, 
pp.241-48 (1978)]. 
Stimulation of the reticuloendothelial system by in vivo administration of 
particulate glucan leads to inhibition of allogenic or xenogenic bone 
marrow graft acceptance in lethally irradiated animals. (See, E. G. Wooles 
and Di Luzio, 1964, Proc. Soc. Exper. Biol. Med. 115: 756-759). This 
finding denotes that glucan will induce host defense mechanisms even 
against normal cells if they are genetically different from the host. 
In addition to effects on reticuloendothelial and immune responses, in vivo 
administration of particulate glucan has been demonstrated to enhance 
hemopoietic activity including granulopoiesis, monocytopoiesis and 
erythropoiesis leading to greater recovery from a lethal dose of whole 
body irradiation (Patchen, 1983, Surv. Immunol. Res. 2: 237-242). 
A number of studies have indicated that in vivo administration of 
particulaee glucan significantly modifies host resistance to a wide 
variety of infectious diseases induced by bacterial, fungal, viral and 
parasitic organisms. In particular, enhanced host resistance to infection 
has been shown when animals are challenged by microorganisms such as 
Eshericheria coli, Staphylococcus aureus, Francisella tularensis, 
Mycobacterium leprae, Streptococcus pneumoniae, Candida albicans, 
Sporotrichum schenckii, as well as viruses such as Venezuelan equine 
encephalomyelitis virus, Rift Valley fever virus, murine hepatitis virus, 
frog virus III, Herpes simplex I and II, and parasites such as Leishmania 
donovani (see review by Di Luzio, 1983, Trends in Pharmacol. Sci. 4: 
344-347 and references cited therein). 
Extensive studies have indicated that particulate glucan has potent 
anti-tumor activity. For example, particulate glucan has been shown to 
inhibit tumor growth and prolong survival in four syngeneic murine tumor 
models including adenocarcinoma BW 10232, anaplastic carcinoma 15091A, 
melanoma B16, and spontaneous lymphocytic leukemia BW5147 (Di Luzio et al, 
1979, in Advances in Experimental Medicine and Biology, Vol. 121A: 
269-290). 
To evaluate the cellular basis of the anti-tumor activity of particulate 
glucan, the anti-tumor cytotoxic activity of peritoneal macrophages, 
derived from control and particulate glucan-treated mice, was studied 
(Mansell and Di Luzio, 1976, in The Macrophage in Neoplasia, Fink, ed. 
Academic Press, New York, pp. 227-243). These studies indicated that 
peritoneal macrophages from guucan-treated mice produced a significant 
cytotoxic response compared to normal macrophages. This observation has 
been confirmed (See, e.g., Barlin et al. 1981,, in Heterogenity of 
Mononuclear Phagocytes, Forster and Landy, eds., Academic Press, New York, 
pp. 243-252) and Chirigos et al., 1978, Cancer Res. 38: 1085-1091). 
Additionally in vitro studies using normal and tumor cells incubated with 
particulate glucan have demonstrated that glucan exerts a direct 
cytostatic effect on sarcoma and melanoma cells and a proliferative effect 
on normal spleen and bone marrow cells (Williams et al., 1985, Hepatology, 
5: 198-206). These studies indicate that glucan, when administered 
therapeutically, will (1) significantly inhibit hepatic metastases; (2) 
inhibit the growth of the primary tumor; and (3) enhance survival, 
possibly by increased Kupffer cell tumoricidal activity as well as by a 
direct cytostatic effect of such glucan on sarcoma cells. 
Notwithstanding these biological properties, the adverse side effects of 
particulate glucans have made these compounds all but useless in clinical 
medicine. 
2.2 ADVERSE SIDE EFFECTS OF TICULATE GLUCANS 
When particulate glucan is administered in vivo to animals, a number of 
severe side effects have become apparent, the most notable being: 
(1) formation of granuloma; 
(2) development of hepatosplenomegaly; 
(3) increased susceptibility to endotoxins; 
(4) activation of complement (anaphylyotoxin); 
(5) development of pulmonary granulomatous vasculitis; 
(6) development of hypotension following intravenous administration; and 
(7) development of microembolism when administered at high concentrations. 
Additionally, there is a relatively high degree of acute toxicity observed 
when particulate glucan is administered in vivo. For example, following a 
single intravenous injection of an aqueous suspension of particulate 
glucan, 20% and 100% morality were observed in mice receiving glucan at 
250 and 500 mg/kg body weight respectively. 
Moreover, due to the particulate nature of the glucan preparation (1-3 
microns), it is difficult to administer via an intravenous route. By way 
of illustration, one patient receiving particulate glucan required 
constant supervision during intraveoous (IV) administration, continuous 
shaking of the IV drip bottle being essential to maintain the particulate 
glucan in suspension to avoid formation of emboli in the patient. 
Although slightly soluble neutral glucans are commerically available, these 
preparations are not suitable for intravenous administration because the 
aqueous solutions have very high viscosity and, more importantly, because 
their use when administered to experimental animals has inevitably been 
accompanied by considerable toxicity. 
Lentinan, a high molecular weight and poorly soluble beta-1,3 and beta-1,6 
glucan obtained from Lentinus edodes, has been studied following 
intravenous administration to dogs. A variety of adverse clinical effects 
were observed following adminstration of lentinan (Ajinomoto Co. Inc., 
Tokyo, Japan) at doses of 2.0, 8.0 and 30 mg/kg/day for 5 weeks. Adverse 
effects included vomiting, erythema, discoloration of the sclera, and 
facial swelling. Circulatory collapse, unsteady gait, altered behavioral 
patterns, excessive salivation were also seen in individual beagles. At 
autopsy, congestion of the gastrointestinal mucosa was observed in animals 
treated with 2.0 or 8.0 mg/kg/day. Morphological changes of liver 
indicated intracytoplasmic material, possibly lentinan, accumulating in 
liver cells. One animal showed circulatory collapse upon the first 
injection at 8.0 mg/kg. While he did recover, the animal experienced 
repeated vomiting episodes with presence of blood indicating hemorrhaging 
of the gastrointestinal tract. Another animal appeared to show a marked 
allergic response, as demonstraeed by erythema and subcutaneous swelling 
(edema) of the face. Autopsy findings demonstrated extensive edema of 
subcutaneous tissue, and congestion of the gastrointestinal tract with 
hemorrhaging. Macrophage cells showed accumulation of material, possibly 
lentinan. (Chesterman et al.,1981, Toxicol. Lett. 987-90) 
Additional toxicity studies were performed in which a variety of doses of 
lentinan ranging from 0.1 to 1.0 mg/kg/da were given intravenously to rats 
for 9 weeks. Toxicity was manifested by the development of cutaneous 
lesions and discoloration of the ears suggesting thromboembolic events. 
(Cozens et al., 1981, Toxicol. Lett. 9: 55-64). 
2.3. UNSUCCESSFUL ATTEMPTS TO SOLUBILIZE TICULATE GLUCANS 
In view of these disadvantages of particulate beta-1,3 glucans for in vivo 
administration, extensive studies were undertaken to develop a soluble 
beta-1,3 polyglucose which might be non toxic, induce no significant 
pathology and yet retain significant immunobiological activity. 
A low molecular weight non-phosphorylated soluble glucan preparation 
prepared by formic acid hydroylsis of particulate glucan has been shown to 
have anti-tumor and anti-staphylococcal activity (Di Luzio et al., 1979, 
Internat'l J. Cancer 24: 7737-779). Unfortunately, the low yield and 
diversity of fractions obtained by this method made this preparation 
non-useful for prophylactic and therapeutic applications. (see Di Luzio, 
1983, Trends in Pharmacological Sciences 4: 344-347). 
Similarly, attempts to solubilize particulate glucan by the addition of 
dimethylsulfoxide (DMSO) a "molecular relaxant" were also unsuccessful. It 
was thought the DMSO would "relax" the triple helical configuration of the 
glucan molecule. However, particulate glucan did not dissolve in the 
presence of DMSO. All attempts to isolate a soluble glucan from the DMSO 
solution resulted in failure. Upon dilution of the DMSO-glucan solution 
with various aqueous media suhh as glucose or saline solutions, the 
particulate glucan was reformed. Following dilution of the DMSO-soluble 
glucan solution with saline, all animals receiving injections of these 
solutions died immediately upon injection due to high concentration of 
DMSO or the reformation of the particulate glucan. Upon precipitation of 
the glucan in DMSO solution by the additon of ethanol (100%), the 
precipitate was collected and lyophilized. When this lyophilized glucan 
was added to water, the particulate glucan reformed. 
Attempts to convert the neutral glucan preparation of particulate glucan to 
a polar-charged preparation by the addition of phosphate or sulfate groups 
as well as by acetylation were also unsuccessful. Each of these procedures 
was conducted following the attempted solubilization of particulate glucan 
by DMSO and in each instance the particulate glucan was reformed. 
3. SUMMARY OF THE INVENTION 
During an exhaustive investigation of methods by which the triple-stranded 
helices of glucan might be "relaxed" sufficiently to permit reaction of 
each of the chains, it was found that when particulate glucan was 
dissolved in a highly polar solvent (such as DMSO) in the presence of a 
strong chaotropic agent (such as urea), the glucan is sufficiently 
structurally disrupted to allow phosphorylation of each of the single 
chains (or strands) such that the resultant phosphorylated glucan shows 
the substantially complete absence of the characteristic triple helica 
structure of particulate glucan. Removal of the resultant phosphorylated 
glucan shows it to be soluble in water, non-toxic, non-immunogenic, 
substantially non-pyrogenic and capable of exerting profound 
immunobiological responses when administered in vivo to animals and 
humans. 
Based on these discoveries, the invention provides a new class of soluble 
phosphorylated glucans (a) in which the poly-[beta-(1-3)glucopryanose] 
chains are phosphorylated in varying degrees; (b) which are non-toxic, 
non-immunogenic, substantially non-pyrogenic, and (c) which are capable of 
exerting pronounced immunobiological responses when administered in vivo 
in animals and humans. These new soluble phopsphorylated glucans, which 
are further characterized by a substantial absence of the triple helical 
structure of particulate glucans, immunostimulate macrophage activity with 
resulting activation of other immunoactive cells in the 
reticuloendothelial and immune systems. Additionally these soluble 
phosphorylated glucans enhance hemopoietic activity including but not 
limited to leukopoiesis. These soluble phosphorylated glucans exhibit 
cytostatic effects against adenocarcinomas and sarcomas in vivo, and 
against lymphocytic leukemia cells in vitro. Not only do these soluble 
phosphorylated glucans stimulate macrophage cells in vivo, but they exert 
profound stimulatory effects on macrophage cells cultured in vitro. Such 
immunostimulttion of macrophage cells is invariably accompanied by 
production of a macrophage cytotoxic/cytostatic factor (MCF), protein or 
proteins of unknown structure, which are selectively toxic to cancers 
cells, particularly adenocarcinomas. 
Additionally, the invention provides a process for producing these soluble 
phosphorylated glucans by dissolving a particulate glucan (preferably 
prepared from Saccharomyces cerevisiae although other microbial sources 
may be used) in a highly polar solvent which contains a strong chaotropic 
agent, and reacting the resultant glucan with phosphoric acid to form a 
soluble phosphorylated glucan, and recovering the resultant phosphorylated 
glucans from the reaction mixture. 
Further, the present invention provides methods and compositions for 
treatment of malignant neoplastic disease in animals and humans which 
comprise administering to an animal or a human a therapeutically effective 
amount of a soluble phosphorylated glucan alone or in combination with an 
anti-cancer or anti-tumor agent. The invention also provides methods and 
compositions for prevention of leukopenia induced by administration of an 
anti-cancer agent which comprise administering to an animal or a human, an 
effective amount of soluble phosphorylated glucan in combination with said 
anti-cancer agent. 
Furthermore, the invention provides methods for stimulating animal and 
human macrophage cells (in vivo or in vitro) to produce and secrete a 
soluble cytotoxic/cytostatic factor (MCF) and the product so produced. 
Specifically, MCF is produced by administering to an animal or a human a 
soluble phosphorylated glucan or by culturing animal or human macrophage 
cells in vitro in culture medium containing soluble phosphorylated glucan. 
The immunobiological properties of the soluble phosphorylated glucans of 
the invention include (1) the ability to prevent mortality due to 
overwhelming gram negative bacterial infections; (2) the ability to 
prevent mortality due to gram positive bacterial infections; (3) the 
ability to modify mortality from spontaneous infections in profoundly 
immuno-suppressed animals and man; (4) the ability to modify enhanced 
susceptibility of immunosuppressed animals and man to gram negative 
bacterial infections; (5) the ability to significantly modify viral 
infections; (6) the ability to modify spontaneous infections induced by 
fungal and other parasitic microorganisms; (7) the ability to 
significantly inhibit primary tumor growth when used alone and to exert a 
synergistic effect against primary tumor growth when used in combination 
with anti-cancer agents; (8) the ability to act synergistically with 
anti-cancer agents in the regression of primary malignant lesions as well 
as metastatic lesions in animals and man. 
Because of these unique immunobiological properties, soluble phosphorylated 
glucan is particularly useful for therapeutic applications against a 
variety of neoplastic conditions as well as against a variety of diseases 
induced by bacteria, viruses, fungi and parasitic organisms. Soluble 
phosphorylated glucan is advantageously administered either alone, with a 
physiologically acceptable pharmaceutical carrier or in combination with 
another bioactive or pharmacological agent and with another therapeutic 
modality such as chemotherapy or surgery.

5. DETAILED DESCRIPTION OF THE INVENTION 
Aqueous soluble phosphorylated glucan represents a novel class of soluble 
phosphorylated glucans in which the poly-[beta-(1-3)glucopyranose] chains 
are phosphorylated in varying degrees. Soluble phosphorylated glucan shows 
the substantially complete absence of the characteristic triple helical 
structure of particulate glucan. Because soluble phosphorylated glucan 
stimulates macrophage phagocytic and secretory activity and increases 
proliferation of macrophages, it is advantageously used to treat malignant 
neoplastic diseases, including but not limited to adenocarcinoma, 
reticulum cell sarcoma, lymphocytic leukemia, melanoma, etc. The soluble 
phosphorylated glucan is advantageously used either alone or in 
combination with another bioactive or pharmacological agent or therapeutic 
modality such as chemotherapy and surgery. 
Because of the potent activity of the soluble phosphorylated glucan in 
stimulating the immune response and reticuloendothelial system, it is also 
particularly useful for prophylactic and therapeutic applications against 
a variety of diseases induced by bacteria, viruses, fungi, and parasitic 
organisms. Copending application of Williams, Browder and DiLuzio, Ser. 
No. 130082, filed on even date herewith is directed specifically to 
compositions and methods for these applications and is incorporated herein 
by reference. 
5.1. PROCESS FOR PREATION OF SOLUBLE PHOSPHORYLATED GLUCAN 
Aqueous soluble phosphorylated glucan is prepared by a process which 
results in a unique class of products different from any other glucans 
previously described. 
Soluble phosphorylated glucan is prepared from particulate glucan, a 
neutral polyglucose deiived, for example from Saccharomyces cerevisiae, as 
follows: particulate glucan is suspended in a solution of a strong 
chaotropic agent in an aprotic solvent such as dimethylsulfoxide (DMSO) 
with constant stirring. The strong chaotropic agent "relaxes" hydrogen 
bonding along the polyglucose chain, thus unfolding the molecule. It is 
preferred to use a fairly high concentration of a strong chaotropic agent 
such as urea ranging from about 4-12M to prevent reformation of hydrogen 
bonds. The mixture is then heated and maintained at about 
50.degree.-150.degree. C. and phosphoric acid is slowly added with 
constant stirring. A precipitate comprising the soluble phosphorylated 
glucan product is apparent after about 1 hour. It is preferred to maintain 
the reaction mixture at about 100.degree. C. for about 3-12 hours to 
increase the yield of bioactive product. In practice, after reaction for 
about 6 hours at about 100.degree. C., the yield is approximately 70-90%. 
The degree of phosphorylation of the soluble product varies slightly with 
reaction time (e.g., 1.48% for 3 hours; 2.23% for 6 hours). 
The bioactive soluble phosphorylated glucan product is isolated from the 
reaction mixture as follows: the mixture is cooled to stop the 
phosphorylation reaction and diluted with a volume of distilled water 
sufficient to resuspend the precipitate. The resulting solution is 
filtered through coarse, medium and fine sintered funnels to remove any 
remaining precipitate. The solution is then molecularly sieved to remove 
all components of less than about 10,000 daltons molecular weight (MW). 
Thus, DMSO, urea, glucose and any unreacted phosphoric acid are removed 
from the solution. Molecular sieving may be accomplished by any method 
that removes these low (i.e., less than about 10,000 daltons) MW 
components. In one illustrative example, the solution is sieved using 
Spectrapor membrane dialysis tubing and dialyzing against running 
distilled water for about 5 days. In another illustrative example, the 
solution is sieved using a Millipore dialyzer/concentrator with a 10,000 
dalton MW membrane filter and a large volume of dialyzing solution. 
Following molecular sieving, the resulting solution is concentrated and 
lyophilized to yield the final soluble phosphorylated glucan in the form 
of a fluffy powder composition. Crystalline structures are not observed. 
The particulate glucan used in the process for preparing the soluble 
phosphorylated glucan according to the present invention may be isolated 
from the cell wall of S. cerevisiae by known methods (see e.g., Di Luzio 
et al., 1979, Internat'l J. Cancer224: 773-779; Hassid et al., 1941, J. 
Amer. Chem. Soc. 63: 295-298 incorporated herein by reference). Briefly, 
in practice the particulate glucan is prepared as follows: dry yeast is 
digested in aqueous sodium hydroxide solution and heated to about 
100.degree. C. for about 4 hours, then allowed to settle overnight. The 
supernatant is decanted and the procedure is repeated three times. The 
residue is acidified using hydrochloric acid, heated to and maintained at 
100.degree. C. for about 4 hours, and cooled overnight. The supernatant is 
decanted and the acid digestion is repeated twice. The residue is then 
washed repeatedly with distilled water and extracted with ethanol for at 
least 24 hours. The reddish-brown supernatant is then aspirated and 
discarded. The ethanol extraction is repeated until the supernatant is 
essentially colorless. The ethanol is removed by repeatedly washing the 
residue with distilled water. The particulate glucan is collected by 
centrifugation or filtration. 
A variety of compounds, other than urea, known to function as "molecular 
relaxants" were also evaluated to prevent reformation of hydrogen bonds 
after DMSO had been used to "relax" the triple helical configuration of 
particulate glucan. These include (1) ethylene diamine tetracetic acid; 
(2) hydrazine sulfate; (3) monoethanol amine; (4) guanidine; (5) guanine, 
and (6) thiourea. Additionally, surfactants and emulsifying agents such as 
Tween-20 and phospholipid emulsifying agents such as Alcolec and Centrolex 
f (lecithin) were also employed in an attempt to solubilize and 
phoshorylate particulate glucan. In no case was a soluble 
immunobiologically active preparation obtained. 
Additionally, soluble phosphorylated glucan can be prepared from neutral 
polyglucose or polyglucose-protein products derived from a variety of 
other microbial sources. A non-exhaustive list of such sources is 
presented in Table 1. 
TABLE 1 
EXAMPLES OF SOURCES OF GLUCAN WHICH CAN BE EMPLOYED FOR THE PREATION OF 
SOLUBLE PHOSPHORYLATED GLUCAN 
Alcaligenes faecalis 
Auricularia auricula-judae 
Auricularia polytricha 
Candida utilis 
Cladosporium fulvum 
Claviceps purpurea 
Cochiliobolus sativus 
Coriolus versicolor 
Corlinellus shiitake 
Corticium vagum 
Grifola umbellata 
Lentinus edodes 
Pichia fermentans 
Poia cocos 
Saccharomyces cerevisiae 
Sclerotium coffeicolum 
Sclerotium delphnii 
Sclerotium glucanium 
Sclerotium rolsfi 
Shizophyllum commune 
Streptococcus salvarius 
Stereum sanguinolentum 
Wingea robertsii 
According to another alternate embodiment of the present invention, the 
soluble phosphorylated glucan may be obtained from the medium used to 
culture an organism such as Sclerotium glucanium using a novel, rapid 
process. Briefly in practice, a colloidal glucan is prepared from 
Sclerotium glucanium as follows: 
A crude sclero-glucan, comprising a polyglucose chain of linearly arrange 
glucose units linked by beta 1-3 glucosidic bonds in which about 30-35% of 
the linear chains have a single glucose unit attached via a beta 1-6 bond 
obtained from the medium used to culture Sclerotium glucanium, is mixed 
slowly with aqueous sodium hydroxide solution with heat and constant 
stirring. The mixture is allowed to stand at room temperature for about 4 
days, heated to about 50.degree.-100.degree. C. for about 15-60 minutes 
and then the mixture is allowed to cool to room temperature. The colloidal 
glucan is isolated from the mixture either by centrifugation or 
filtration. To illustrate, when filtration is used a series of filters of 
decreasing pore size such as 3.0, 1.2, 0.8, 0.65 microns are used. The 
resulting dark amber filtrate is diluted with water and molecularly sieved 
to removed all components of less than about 10,000 daltons molecular 
weight. In one illustrative example, the mixture is sieved using 
spectropor membrane dialysis tubing and dialyzing against 40 liters of 
water. The final concentrated mixture having about neutral pH is 
lyophilized yielding an amber spongy glucan material. In another 
illustrative example, the mixture is dialyzed and concentrated using a 
Pellicon dialyzing unit. The mixture is dialyzed against 75 liters of pure 
water. The final concentrated mixture having about neutral pH is 
lyophilized yielding an amber spongy colloidal glucan material. 
Soluble phosphorylated glucan is prepared from the colloidal glucan as 
described above. Briefly, colloidal glucan is suspended in a solution of a 
strong chaotropic agent in an aprotic solvent such as dimethylsulfoxide 
(DMSO) with constant stirring. The strong chaotropic agent "relaxes" 
hydrogen bonding along the polyglucose chain, thus unfolding the molecule. 
It is preferred to use a fairly high concentration of a strong chaotropic 
agent such as urea ranging from about 4-12M to prevent reformation of 
hydoogen bonds. The mixture is then heated and maintained at about 
50.degree.-150.degree. C. and phosphoric acid is slowly added with 
constant stirring. A precipitate comprising the soluble phosphorylated 
glucan product is apparent after about 1 hour. It is preferred to maintain 
the reaction mixture at about 100.degree. C. for about 3-12 hours to 
increase the yield of bioactive product. In practice, after reaction for 
about 6 hours at about 100.degree. C., the yield is approximately 70-90%. 
The degree of phosphorylation of the soluble product varies slightly with 
reaction time (e.g., 1.48% for 3 hours; 2.23% for 6 hours). 
5.2. CHARACTERIZATION OF SOLUBLE PHOSPHORYLATED GLUCAN 
The solubility of the soluble phosphorylated glucan obtained from S. 
cerevisiae prepared according to the present invention is greater than 
about 50 mg/ml in water. Aqueous solutions of the soluble phosphorylated 
glucan are non-viscous and do not taste sweet. 
5.2.1. ELEMENTAL COMPOSITION 
The elemental composition of the soluble glucan preparation, determined by 
Galbraith Laboratories, (Knoxville, Tenn.) is illustrated in Table 2. The 
data presented in Table 2 permits the average empirical formula of this 
preparation to be writtnn as follows: 
EQU C.sub.40 H.sub.87 PO.sub.37. 
Thus, there is an average of one phosphate group for every 6.6 glucose 
residues in the soluble phosphorylated glucan. 
TABLE 2 
______________________________________ 
ELEMENTAL COMPOSITION OF 
SOLUBLE PHOSPHORYLATED GLUCAN.sup.a 
Element or Component 
Mole % 
______________________________________ 
Carbon 34.66 
Hydrogen 6.29 
Oxygen 42.83 
Nitrogen 0.64 
Sulfur 0.11 
Phosphorus 2.23 
Water of Hydration 11.78 
______________________________________ 
.sup.a Determined after 6 hours phosphorylation. 
5.2.2. STRUCTURAL CONFIGURATION 
A number of methods were utilized to determine the molecular weight (MW) 
and various features of the structural configuration of the soluble 
phosphorylated glucan. 
5.2.2.1. MOLECULAR SIEVING 
Column chromatography using Sepharose CL-6B-200 (Pharmacia Fine Chemicals, 
Piscataway, N.J.) indicated that 80% of the soluble glucan has a MW range 
from 10,000 to 100,000 daltons, while 20% has a MW range from about 
100,000 to about 500,000 daltons. 
5 2 2.2. NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY 
Carbon-13 nuclear magnetic resonance (.sup.13 C-NMR) spectroscopy using a 
Brucker WP-200 spectrometer (Brucker Instruments, Billerica, Mass.) was 
performed to determine several structural properties of the soluble 
phosphorylated glucan from S. cervisiae prepared according to the present 
invention. 
The samples for NMR studies were prepared using a 10% deuterium oxide 
(D.sub.2 O) in water. Samples were first run with no reference material, 
and then after the addition of a small aliquot of 1,4-dioxane. All samples 
were placed in 10 mm diameter tubes. 
The .sup.13 C-NMR spectral study of soluble phosphorylated glucan indicated 
a beta-1-3 glucan structure with no branching at the C-6 carbon (see FIG. 
1). The NMR spectrum indicates a substantial absence of the triple helical 
structure of particulate glucans. The degree of phosphorylation was 
estimated to be 3.6% which is in essential accord with analytical data. 
In contrast to the spectra of soluble phosphorylated glucan from S. 
cerevisiae prepared according to the present invention, a lentinan 
preparation (Ajinomoto Co. Inc., Tokyo Japan), a branched beta-1-3 and 1-6 
D glucan, at either 40 mg/ml (FIG. 2A) or 3 mg/ml (FIG. 2B) demonstrated 
attenuation of the NMR spectra. This is presumed to be due to the gel 
state of this molecule, particularly at 40 mg/ml concentration. No signals 
were obtained in a 10% D.sub.2 O solution in the non-gel 3 mg/ml 
concentration. 
Comparison of FIG. 1 with FIG. 2 demonstrates complete structural and 
conformational differences between lentinan and soluble phosphorylated 
glucan. In contrast to the disordered conformation of lentinan at the 
beta-1-6 linkages (Saito et al., 1977, Carbohydrate Research, 58: 293-305) 
ordered conformation of soluble phosphorylated glucan according to the 
present invention is manifested. 
5.3. NON-TOXlClTY, NON-PYROGENICITY NON-IMMUNOGENICITY OF SOLUBLE 
PHOSPHORYLATED GLUCAN 
Since the soluble phosphorylated glucan offers important advantages over 
particulate glucan as an injectable biological response modulator, 
characteristic toxicity, pyrogenicity and immunogenicity of the soluble 
glucan are described below with particular reference to comparison of 
these properties of particulate glucan. 
5.3.1 NON-TOXICITY 
Acute toxicity as evaluated following a single intravenous injection of 
soluble phosphorylated glucan at a variety of doses into normal animals. 
Treated animals were observed for 30 days post-injection. 
In one series of experiments, 49 ICR/HSD mice were divided into 3 groups of 
15 mice each and 2 groups of 2 mice each. Groups 1-3 received 0.5 ml 
saline solution containing soluble phosphorylated glucan at respectively 
40, 200 and 1000 mg/kg; Groups 4 and 5, 1600 and 2000 mg/kg. No mortality 
was observed in any group. Moreover, no physiological or behavioral 
alterations were apparent. In marked contrast, in mice treated similarly 
with particulate glucan, 20% mortality was observed at 250 mg/kg and 100% 
mortality at 500 mg/kg. 
In another series of experiments, two groups of 5 Sprague Dawley rats each 
were treated with soluble phosphorylated glucan at respectively 250 and 
500 mg/kg via intravenous injection. No mortality or alteration of 
physiological or behavioral functions was apparent in either group. In 
contrast, 30% and 100% mortality were observed following intravenous 
injection of particulate glucan at 75 and 150 mg/kg respectively. 
Chronic toxicity was evaluated following twice weekly intravenous 
injections of saline solution containing soluble phosphorylated glucan at 
0, 40, 200 and 1000 mg/kg doses. Body and organ weights, gross and 
microscopic pathology, serum electrolytes, solutes and serum enzymes 
indicative of renal and hepatic function were monitored. 
In one series of experiments mice were weighed respectively at 0, 8, 11, 
15, 22 and 30 days post-treatment With soluble phosphorylated glucan. No 
significant difference was observed in body weight at any dose of soluble 
phosphorylated glucan administered. After 30 days chronic treatment, 
animals were sacrificed. No change was seen in weight of liver, lung and 
kidney. A statistically significant increase in spleen weight was noted in 
mice treated with 40 and 100 mg/kg soluble glucan (0.01&lt;p&lt;0.001), but not 
in mice treated with 200 mg/kg. 
In another series of experiments, mice were weighed respectively at 0, 15, 
30, 49 and 60 days post-treatment (twice weekly) with soluble 
phosphorylated glucan. No significant difference was observed in body 
weight at any dose of soluble phosphorylated glucan administered. After 60 
days chronic treatment with soluble phosphorylated glucan, animals were 
sacrificed. No significant difference was observed in weight of the liver, 
kidney or lung. A statistically significant increase in spleen weight was 
apparent in mice treated with 1000 mg/kg soluble phosphorylated glucan 
(p&lt;0.001). 
After 30 or 60 days chronic treatment, no significant alteration was 
apparent in the following serum components: glucose, blood urea nitiogen 
(BUN), uric acid, cholesterol, triglycerides, total protein, albumin, 
globulin, creatinine, calcium, phosphorous, sodium, potassium, chloride, 
bicarbonate and anion gap. Moreover, no significant alteration was 
apparent in the following enzymes: alkaline phosphatase, lactic 
dehydrogenase, serum glutamic oxalacetic transaminase, serum glutamic 
pyruvic transaminase and creatinine phosphokinase. No change was 
detectable in serum bilirubin. 
Histological studies on tissues obtained from mice following 30 days 
chronic treatment showed essentially normal liver histology in mice 
receiving 40 and 200 mg/kg soluble phosphorylated glucan per injection. In 
animals receiving 1000 mg/kg soluble phosphorylated glucan, monocytic 
infiltrates were readily apparent in the liver. Lung and kidney tissues 
were essentially normal in all mice. 
Histological studies on tissues obtained from mice following 60 days 
chronic treatment showed few hepatic granuloma of an isolated nature in 
animals receiving injections at 40 and 200 mg/kg doses. A higher number of 
monocytic infiltrates was observed in mice receiving injections at 1000 
mg/kg. In all autopsied animals, lung tissue was essentially normal. 
Chronic toxicity was further evaluated using guinea pigs (Harlan Sprague 
Dawley, Houston, Tex.) receiving 5 ml intraperitoneal injections of saline 
solution containing soluble phosphorylated glucan at 250 mg/kg for 7 days 
(FDA required test). Results presented in Table 3 indicate that there was 
an impairment of growth of guinea pigs receiving soluble glucan treatment 
when compared to controls receiving an equivalent volume of 0.9% saline 
solution. Following 7 days chronic treatment, body weight of treated 
animals was, however, significantly increased by 9% as compared to initial 
weight. 
TABLE 3 
__________________________________________________________________________ 
EFFECT OF CHRONIC ADMINISTRATION OF 
SOLUBLE PHOSPHORYLATED GLUCAN 
ON BODY WEIGHT 
Mean Body Weight (gm).sup.a 
Treat- 
Days 
ment 1 2 3 4 5 6 7 
__________________________________________________________________________ 
Saline 
213.8 .+-. 4.0 
225.2 .+-. 6.6 
227.4 .+-. 7.1 
234.2 .+-. 7.3 
239.7 .+-. 6.1 
244.2 .+-. 5.2 
253.8 .+-. 7.5 
SPGLN.sup.b 
208.9 .+-. 2.9 
202.3 .+-. 5.0 
203.9 .+-. 5.3 
207.4 .+-. 5.3 
214.3 .+-. 6.0 
215.6 .+-. 6.6 
227.3 .+-. 6.6* 
__________________________________________________________________________ 
.sup.a Values represent mean body weight (gm) .+-. standard error. N = 5 
animals. 
.sup.b SPGLN designates soluble phosphorylated glucan. 
*p &lt; 0.01 
Chronic Toxicity was also evaluated in 2 adult female dogs receiving twice 
weekly intravenous administration (5 mg/kg) of soluble phosphorylated 
glucan for 120 days. The dogs were fed Purina Chow and water ad libitum 
supplemented with one can commercial dog food (Alpo.TM.) twice weekly. 
Body weight and serum solutes, electrolytes and enzymes were monitored at 
0, 17, 24, 38, 80 and 120 days. Following 120 days chronic treatment, a 
mean weight gain of 2.8 kg or about 22% body weight was observed. 
No significant difference was observed in the following serum solutes: 
glucose, BUN, uric acid, cholesterol, triglycerides, total protein, 
albumin, globulin, or creatinine. No significant difference was observed 
in the following serum electrolytes: calcium, phosphorous, sodium, 
potassium, chloride, bicarbonate, and anion gap. No significant difference 
was observed in the following serum enzymes: alkaline phosphatase, lactic 
dehydrogenase, serum glutamic oxalacetic transaminase, serum glutamic 
pyruvic transaminase and creatinine phosphokinase. 
Additionally, no significant difference has been observed in the serum 
biochemistry of a patient following therapy for 3 months with soluble 
phosphorylated glucan at 50 mg/ml, administered three times per week. 
5.3.2. NON-PYROGENICITY 
Pyrogenicity of soluble phosphorylated glucan was evaluated following a 
single intravenous injection to conscious dogs at doses of 7.5 mg/kg and 
30 mg/kg. Body temperature was monitored for 14 days post-injection. 
Results presetted in Table 4, demonstrate no pyrogenic reaction in this 
chronic animal model. 
TABLE 4 
______________________________________ 
ABSENCE OF AN ACUTE OR CHRONIC 
PYROGENIC RESPONSE IN DOGS 
Mean Body Temperature (.degree.C.) 
Treatment Dose.sup.a 
Time (Hours) 
(mg/kg) 0 1 6 24 144 336 
______________________________________ 
7.5 38.3 37.4 38.3 38.3 38.3 38.4 
30.0 38.6 37.0 38.0 38.5 38.4 38.6 
______________________________________ 
.sup.a N = 3 dogs/group. 
Pyrogenicity was also evaluated using three dogs anesthetized with Nembutal 
(30 mg/kg) receiving multiple injections of increasing doses of 1, 5, 10, 
15, 25 and 50 mg/kg of soluble phosphorylated glucan over a three hour 
period. Body temperature was determined at 15 minutes following bolus 
injections. No pyrogenic effect was observed at any dose. 
Pyrogenicity of soluble phosphorylated glucan was also evaluated in 
rabbits. Seven rabbits were divided into 2 groups of 2 and 5 rabbits each. 
Group 1 received an isovolumetric saline solution; Group 2, received 5 
mg/kg soluble phosphorylated glucan in saline solution by intravenous 
injection. Core body temperature was monitored at 15 minute intervals for 
100 minutes following a single bolus injection. Control rabbits showed a 
mild decrease of 0.2.degree. C. in body temperature. Rabbits treated with 
soluble phosphorylated glucan showed a mean increase of 0.44.degree. C. 
Thus, there was a slight pyrogenicity seen in rabbits. 
5.3.3. NON IMMUNOGENICITY 
The interfacial ring test, designed to detect the presence of IgG 
antibodies, was used to evaluate the immunogenicity of soluble 
phosphorylated glucan when chronically administered to dogs for 120 days. 
Serum samples were obtained from an adult female dog following 120 days 
chronic treatment with twice weekly intravenous injections of 5 mg/kg 
sterile, pyrogen-free soluble phosphorylated glucan. The interfacial ring 
precipitin test was performed as follows 0.1 ml of undiluted antisera was 
pipetted into test tubes. The antigen or phosphorylated soluble glucan at 
dilutions of 1:2, 1:4, 1:8,.1:16 and 1:32 was layered onto the anti-sera 
to form straight interface. Formation of a white precipitin ring at the 
interface indicates the presence of antibody specific for the glucan. No 
precipitin ring was detected at any antigen dilution. 
5.4. USES OF SOLUBLE PHOSPHORYLATED GLUCAN 
Because soluble phosphorylated glucan influences very fundamental host 
defense systems of the body regulating the number, functional activity and 
interaction of macrophages, T and B lymphocytes, leukocytes and natural 
killer cells as well as their humoral and secretory components, it 
possesses the potential for non-specifically modifying an extensive array 
of diseases including infectious and neoplastic diseases. 
Soluble phosphorylated glucan demonstrates a number of characteristics 
which make it particularly advantageous for the prohpylactic and 
therapeutic treatment of neoplasms including, but not limited to the 
following advantages: 
(1) Soluble phosphorylated glucan exerts direct inhibitory effects on the 
proliferation of tumor cells including, but not limited to lymphocytic 
leukemic cells, melanoma cells and reticulum cell sarcoma cells; 
(2) Soluble phosphorylated glucan has very low toxicity; 
(3) Soluble phosphorylated glucan prevents and corrects the development of 
leukopenia; 
(4) Soluble phosphorylated glucan enhances a variety of diverse aspects of 
cellular and humoral immune responses of the host; and 
(5) Soluble phosphorylated glucan prevents or reverses the development of 
immunosuppression in the host. 
Due to the stimulation of macrophage phagocytic and secretory activity and 
increased proliferation of macrophages caused by soluble phosphorylated 
glucan, this composition can advantageously be used either alone or in 
combination with other modalities such as surgery and chemotherapy, to 
treat malignant neoplastic diseases including, but not limited to 
adenocarcinoma, reticulum cell sarcoma, melanoma, lymphocytic leukemia, 
etc. 
Soluble phosphorylated glucan has additive or synergistic effects when used 
in combination with a broad range of antitumor or anticancer agents. 
Additionally soluble phosphorylated glucan is effective in preventing 
and/or correcting the development of leukopenia which is often associated 
with the use of a variety of antitumor or anticancer agents. Table 5 
illustrates some of the antitumor or anticancer agents that may be used in 
combination with soluble phosphorylated glucan. Table 5 is in no way 
intended to be an exhaustive list. Additionally, a combination of one or 
more antitumor or anticancer agents may be used together with soluble 
phosphorylated glucan. 
TABLE 5 
EXAMPLES OF ANTI-TUMOR AGENTS WHICH MAY BE COMBINED WITH SOLUBLE 
PHOSPHORYLATED GLUCAN FOR TREATMENT OF NEOPLASIAS 
I. ALKYLATING AGENTS