CA2141845A1 - Improved method for preparing soluble glucans - Google Patents

Abstract

2141845 9403498 PCTABS00030 A highly efficient, rapid method for preparing aqueous soluble glucans is described.

Description

2 1 ~
```;~ W094f03498 . Pcr!usg3/o7329 ~- .

IMPROVED METHOD FQR PREP~I~G SOLUBLE GLUC~S ~ `.
t l. FIELD OF THE I ~ ENTION
This invention relates to an:improved, , `.
5 highly e~ficient method for preparlng~soluble`glucans. ' .
The method advantageously avoids the use of a highly : `
polar solvent such as dlmethyl sulfoxide:(D1~SO). The soluble glucans~prepared by the~method of tne~
invention are non~toxic and:exer:t pronounced ~ -.
; lO i~ unobiologica~l~.responses when administered~ln vivo, :~ ; most ~notably i ~ unostimulatlon~of~màcrophage~
activities and~stimul~ation~of hema;topoiet:lc~bone~
marrow~activity.`~The~soluble glucans à~lso exhibi~
significant effects against;~mali~nant neoplasms~
lS including melanomas~and~sarcomas.

: 2. ~BACKGRO ~ D~O~F THE I ~ NTION
The tèrm:~"glucan" refers generically to~a ;
variety~of~naturally~occurring~homopolysaccharldes~:or 20~ polyglucoses, includ~lng:po:l~ ers~:such~:as cellulose,~
amylose,~glycogen,~;lamina~rians, st~arch~ e~c.~ Gluca~
enco asses bran h d~and~unbra che~d~cha:ins;o~:glucose~
. uni:~s linked:by 1-:3,~ 4,~and l-6~ glucosid:ic~:bonds~
that~may~be of either th~e~alpha or~beta;~type~
2~5~ As defi;ne:d`h } n,~'particu ~te~g:lucan"~
deslgnatès a~watèr-insoluble~particulàte~(`abqut~ 3 .po~lyglucose such~ rived~ : th~e.cel;l~wal of~
the yeas:t SaccharomYces cerèvis~iae.~Particulate~
glucan~is,;~acromolecùlar andjcompr~ ses~q~ clqsed1chain .;

3~ of ~ glucopyranose.units~united by~a~series~of~ l-3 glucosldid~lin~ages ~( assid e al l94l:,~ m r.

^àncar 24:773-7:~9).~ ra~y~ ra on~s~udi s h ve~
de~ nstrat`~; hat~:p ~ ~ic a 2141~llà ~

WO 9~/03498 PCr/,US93/q732g ':`"` i " ` ` ' form~of a triple-stranded helix. (Sarko et al., 1983, - `~
Biochem. Soc. Trans.~ 13s-142). `~
Particulate glucan is a potent activator of ~`
the macrophage/monocyte cell series, complement, as 5 well as T and B cell lymphocytes. Thus, particulate :~
glucan has profound e~fects on both the reticuloendothelial and immune systems. Particulate `;
glucan has been shown to modify host resistance to a wide variety of infectious diseases (see review by DiLuzio, 1~83, Trends in Pharmacol. Sci. 4:344~347 and references cited t~erein). ~In addition particulate glucan has been shown to inhibit tumor growth~and prolong survival in syngeneic murine tumor models (DiLuzio et al.,~ 1979, Advances in Exp. Med.~Biol. ' ~
121A:269-290). In vitro studies using normal and ~ ```;
tumor cells incubated w~ith particulate glùcan have demonstrated that glucan exerts a cytostatic effect on ,~
sarcoma and melanoma cells and a proliferati~e effect ~ ;~
on normal spleen and bone~marrow cells (Williams et al., 1985, Hepatology 5:198-206).
Notwithstanqing the ~eneficial biological ~ ,`;
properties of particulate`glucans,~the a~dverse side effects of particulate glucans have made these compounds all but useless in clinical medicine. When particulate glucan is~administered~In vivo to animals, a number of severe~side;effects have~become apparent, the~most notable being: (1) formati;on of~granuloma (sarcoidosis); (2j development of hepatosplenomegaly;
increased~su$ceptibility to gram negative~
infections and endotoxins; (4;) activation of comp~lement (anaphy~latoxin); (5~) developmen~ of~
pulmonary granulomatous~vasculit~1s~ (6) development of hypotension fo11Owing lntravenous admlnlstration; and ; ~ ~ 3 (7) development of microembolism when~administered at high concentrations.

'"'""' W094~0~98 ; ~141~1S PCr/U~g3l07329 -3- 1' .,~
~ ` Additionally, there is a relatively high degree of acute toxicity observed when particulate ~
glucan is administered ln vivo. For example, ~ ';`
following a single intravenous injection of an aqueous ' `' suspension of particulate glucan, 20% and 100%
mortality were observed in~ mlce receiving glucan at -`
250 and 500 mg/kg body weight respectively.
Moreover, due~to the particulate nature of the g~ucan preparation~ 3 ~ it is~difficult to administer via'an;intravenous route.~By way o~
illustratian, one~patient recei~in~ particulate glucan~
required constant superv;ision`~during intravenous~(IV)~
administration, continuous~shaking of the IV~drip bottle being essential to maintain;the particulate ~5 glucan in suspens~ion~to~avoid formation~o~f~emboli in the patient.
Although slig~htly;~soluble;neutral~glucans~
are~commercially~ava~il`able,~these preparations are not~
su~itable~for'~intravenous~administration~because the 20 aqueous solutions~have~ very high vi~scosity~and, more ~ Lj~
; ; importantly, because;~their use~when administered to experimental animals~has~inevitably~beèn accompanied by c;o~siderable~toxi~city'.
In view~of~the disadvantages of~particulate~
2~S ~-1,3 glucans for ln vivo administrat`ion,~lextensive `~
`studies~were~previcusly~und~ertaken~:~to~develop~a soluble ~-l,3 pclyg1ucose~which~might~be~`~non-toxic~ x'}' induce no signi~icant pathology, and~yet~retain ;ignificant~immunobiological~a~t ~ A ~low~molecular~weight~non-phosphoryla'ed~
soluble glucan~p~re ation~p ep;ar ~ formi acid~
hyd`olysi~s~of~p ~¢ ate~glucan~'h~as~ ~en~sh n~to~
have`anti~- ~ mor~and~ant~i-s~aphylococ l;'a ' ty~
`(Di~ zio~et~ al.~ 9~9,'~Internat~'l J~ C ce~ 773~
3 ~ unfor-unae ~-he~ y ~ sity~or~ 3 2 1 4 1 ~3 W094/03498 PCT/~S~3/07329 . .

fractions obtained by this method made this preparation non-useful for prophylactic and therapeutic applications. (See DiLuzio~, 19~3, TrendS
in Pharmacological Sciences 4:344-347).
Similarl~, attempts to solubiliæe `
particulate glucan by the addition of ` ~.
dimethylsulfoxide (DMS0) a "molecular relaxant" were `~
also unsuccessful. It was thought the D~SO would "relax" the triple~helical configuration of the glucan 10 molecule. Indeed, particulate glucan dissolves in the presence of DMS0. All attempts to isolate a soluble glùcan from the DMS0 solution, however, resulted in failure. Upon dilution of the DMS0-glucan solution with various aqueous media such 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 20 particulate glucan. Upon précipitation of the glucan ~
` in DMSO solution by the addition of ethanol (100%), ~`
the precipitate was collected and lyophilized. When ` ;
this lyophilized glucan was added to water, the particulate ~lucan reformed.
Early attempts to convert the neutral ~lucan ,, preparation of particulate glucan to a polar-charged preparation by the addition of phosphate or sul~ate groups as well as by acetylation were also uns,uccessful. ;Each of these proced;ures~was conducted following the so}ubi}ization of particulate glucan by DMSO and in each instance the partlculate glucan was reformed. ~ ?~ ~;
A neutr~l preparation of particulate slucan was successfully converted~lnto a ;stable~solubllized~;
35 form termed "soluble phosphorylated glucan" ` ~ `~

`:

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~`WO 94/03498 ` PCI/~JS93/~17329 1 `~

-5- ! :`

(hereinafter termed "glucan phosphate") through phosphoric acid hydrolysis using the method described briefly below As de~ined herein, the term "glucan phosphate" or "soluble phosphory~ated glucan" ref-ers 5 to the class of glucans solubilized by the addition of charged phosphate groups through reaction with ~
phosphoric acid These are the same or substantially ```
similar to those substances as described in U~S
Patents Nos 4,739,046, 4,761,402; 4,818,752 and ``
10 4,833,131 This solu})le phosphorylated glucan ~is non~
toxic, non-immunogenic, and substantial~y non-pyrogenic (see U S Patent Nos 4,739,046; 4,761,4Q2;
4,818,752 and 4,833,131) According to the method of U S Patent No 15 4,739,046, glucan phosphate was prepared as follows ~ i particulate glucan or a polyglucose-protein complex wa~s suspended in the highly polar aprotic solverlt ~``x DMS0 A strong chaotropic agent, urea, was added, the mixture heated and maintained at 50-150C with~
20 Constant stirring while phosphoric acid was slowly `
added Prefera~ly, the reaction mixture~ was malntained at about 100C for about 3-1~ hours to increase the yield of the; bioactive product The `
product was isolated and the DMSO, urea,~glùcose, and 25 any unreacted phosphoric acid were removed The yield, after reaction~f~or about 6 hours at 100C, is stated~ to be about~ 70-90% ~ ?,~
Another~new class of soluble glucans, ~in whi~h the polyglucopyranouse chalns~have acqulred~ !a 30 charged group from a non-phosphorous containin~
hydrolytic acid, are described ln~ copending ;
applicatlon Serial No 07/649,5~27 `~T~he~ soluble glucans~ havlng a charged~ group, including;~such~c s ~a ~ ;
sulfate or nitrate group, are also ~c~apabl~e of exerting 35 a pronounced Immunobiolo ~cal e~ec-~when adm_n~stered~

21418'1~
W094/0~ PCT/US93/0732 in vivo. llhese soluble glucans immunostimulate macrophage activity with resulting activation of the immunoactive cells in the reticuloendothelial and immune systems. In addition, these soluble glucans enhance hematopoietic bone marrow activity.
According to the methods of Application Serial No. 07/649,527, the soluble ~luc~ns were `
prepared as follows: particulate glucan was suspended in a solution of DMSO and urea. The concentrated mi~ture was heated to about 50-150C, a concentrated hydrolytic acid, such as sulfuric or nitric acid alone or in the presence of DMS0 was added and the reaction mixture was maintained at about 50-150C with stirring. The bioactive product was isolated and the , . . .
DMSO, urea, glucose and any unreacted aci~were removed. After about 6 hours at 100C, the hydrolytic `
acid was used alone, the yield i5 stated to be about 37.5%, when the hydrolytic acid was added with additional DMS0, the yield is stated to be about 98%.
U.S. Patent No. 4,707,471 describes a water-soluble aminated ~ 3 bound D-~lucan composition and a method for preparing such composition. According to one embodiment of the method, ~ 1,3-D-glucan, preferably curdlan or laminarian was hy~rolyzed in 90~ ;
formic acid. The acid was removed by evaporation, water was added and the mixture was refluxed~for an hour. The mixture~ was then separated on a Sephadex G- ~ ;
50 column and the highest molecular weight fraction recovered and further reacted as follows. l'~he~
hydroly~ad glucan was dissolved in water containing bromine at pH 7 and allowed to stand~until all the bromine was consumed (24-48 hours).~ The pH was~ . 7, adjusted to 5.0 and the mixt~re dialyzed;agalnst water ~-and freeze dried. The oxidized glucan was added to a .
solution of ammonium acetate or 1,6-diaminohexane~in .
.

~v~ 2 1 4 1 ,~
`` W094/03498 PCT/US93/07329 l:7 ' 1 `
', `. `'`'```
wate~ adjusted to pH 7.0 with acetic acid together ! : `
with sodium cyanoborohydride and allowed to stand for I `
7 days with stirring. The aminated glucan was obtained after dialysis and freeze drying. In another 5 embodiment of the method, the hydrolyzed laminarian ``
was dissolved in DMSO prior to acetylation and then l~`
aminated as described above.
Wosl/o349s by Jamas describes soluble ``
preparations of neutral glucan polymers by a method involving treatment of glucan particles with a "unique sequence of acid and alkaline treatments." Whole glucan particles were suspended in acid solution, generally about pH 1-5 at 20-100C, preferabl~y using ;~,an organic acid~such as acetic or formic acid. The `
acid insoluble glucan was removed and the pH adjusted to pH 7-14. The slurry was resuspended in hot alkali, such as NaOH or KOH at 0.1-10 N at 4-120CC. The soluble glucan was recovered and further purified.
U.S. Patent No. 3,883,505 describes a method for improving solubility of poorly soluble or water-insoluble polysaccharides such as pachyman, explain, lentinan, etc., using strong, hot aqueous~solutions of urea, thiourea, guanidine and their N-lower alkyl deriYatives.
U.S. Patent Nos. 3,987,166 and 3,943,247 describe, respectively, treatment~of animal tumors and prevention and treatmen~ of bacterial~infections. As , indicated, completely unlike the soluble phosphorylated glucans prepared by the present method 30 which are non-viscous, the glucans of these patents ~ ;
are highly viscous and difficult to~prepare in ~aqueous solution of higher concentrations~ than~0~5~ aqueous solution Notwithstanding the above methods, there 3S still remains a need for high efficiency, faster :: ~ ~ .;

2 1 ~
W094/034~8 PCT/US93/07329 ?; `: :
, i I ` ~ "

: ' methods for obtaining bioactive soluble glucans which -can be used as hiological response modifiers. The present method meets this long felt need. ~ ~;
:, S 3. SUMMARY OF THE PRESENT IN~ENTION
The present invention provides an improved, highly efflcient method for preparing aqueous~soluble glucans. The method avoids the use of DMSO.
The method o~ thP invention for preparing a `
soluble glucaIl, comprises the steps of:
(a) mixing a neutral polyglucose or a polyglucose protein complex with a strong chaotropic reagent and grinding the mixture to form a ~ine ~ :
powder; `
~5 (b) reacting the fine powder mixture with a strong solution of concentrated phosphoric acid -to form a soluble glucan and recovering the resultant ~ I;
soluble glucan from the mixture.
,:' `', 2 0 ` 4 . BRIEF DES~CRIPTION OF THE FIGURES
The present invention may he more fully understood by reference to the following detailed ~ ~`
description o~ thé invention, exampL~es of specific embodiments of the invention and the~appended figures ~;~
in which~
FIG. l~ B)~lllustrates~helica1 coil transiti~n analyses~ Dextran (70kD) (~-~) ser~ed as the linear control. Congo Red in~sodium hydroxide ~o~
! p), j served as the~nega'ive control~ FIG~ ,is the ~` 30 helical coil transition analysis for~soluble ;~ pbosphorylated~ glucan prepared accord~ing~to the~mathod ; o~ the present invention. ;FIG. lB~is~the helical coil transition analysis for soluble,~pho~phoryla~ed ~lucan~
prepared according to the prior ar~method.

2 1 4 1 ~ 4 ti ~" W094/03498 PCT~US93/07329 ~`
_9_ 1-` ``

~ FIG. 2 (A-B) illustrates 13C-NMR spectra of soluble phosphorylated glucan. FIG. 2A shows the NMR
spectrum of soluble phosphorylated glucan prepared according to the prior art method. FIG. 2B shows~the ~ -NMR spectrum of soluble phosphorylated glucan prepared according to the method of the present invention.
:' 5. ~ ```~

5.1 PROCESS FOR PREPARING SO~UBLE GLUCANS '~
According~ ~o éhe method of the present invention, aqueous soluble`glucan is~prepared from a neutral polyglucose or polyglucose-protein c~omplex obtained from a variety of microbial sources as lS follows: a neutral polyglucose or a~ polyglucose-protein complex is mixed with a strong chaotropic agent,; such as urea, and the dry mixture is thoroughly mixed and ground into a fine powder.~ Any means of grind;ing to form a fine~powder is suitable~.~ For preparation of small batches, a mortar~and~pestle is satisactory. In practice, about 1-4 gm of neutral polyglucan or a polyglucose-protein-complex is mixed with about 10-20 gm of~urea. ;Co~ncent`rated~phosphoric acid, about 5-50~ml~(concentrated,~about Z0~43~) is ; ~`~
25~ added to form a slùrry~and~the reaction mixture is heated to about 6~0-80C~with~cons~tant~stirrlng. The~
rèaation mixture is~maintained at about~60-8~0C for l~

6 hours untiI a precipitàte comprising the~soluble ~
phosphorylated glucan forms. Abou~ilO ml~of~d1stilledl ~l `i 30 water is added to~reform a slurry ~and the~reaction ~ ,7;
mixture maintained a~`about~60-sooC~with`stirring.
The addition of water~is repeated s~èver~al times,; ~
preferably about three times, to mainta;ir.~`~a slurry~and ~ heating is con~inued. It~is~preferable~to~`maintain~
; 35 the reaction mixture at~about 6~0-80C~for~àbout~1-2 2 1 ~
WO9~/~98 PCT/US93/07329'~

.
hours. In practice, after reaction for about two hours, the yield is about 97%. During the reaction, ammonia is released from the urea and the smell of '~
ammonia is most noticeable between about 1-2 hours r ~, 5 after heating is begun. - ' In one embodiment, about 1-4 gm of particulate glucan is mixed with about 10-20 gm of urea and about 20-25 ml of concentrated phosphoric acid is used to form the slurry which is treated as ;' 10 above. ~ -~
The soluble phosphorylated glucan lS ;;
isolated from the reaction mixture as follows: the , ,' mixture is removed from the heat and dissolved~in a ~,, large volume of distilled water so that the , -15 precipitate is resusp nded. The resulting solution is '~
filtered through coarse,~medium and fine sintered filters to remove any remaininq precipitate. The ~ .;
solution is then molecularly sieved to~remove all co~ponents of less than 10,000 MW. ~Accordingly, any 20 urea, glucose and unreact~d phosphoric~;acid are ''' remo~ed from the solution. Any sui~table method known in the art for molecular sieving can be used~ For ' ~,~
example, the solution can be sieved u5ing Spectrapor , "
membrane dialysis tubing dialyzed against;running `~
2S di8tilled water. In another example, the~solution can ,', be~sieved using a Millipore dialyz~er/concentrator with ~, a 10,000 dalton MW membrane filter~,and a larger volume of dialyzing solution.
The method of the present,~invention is,more~
30 rapid and more efficient than the prior art method for , "
preparing~soluble gIucan phosphate.;~ The time required~
for solubilization is redu~ed,~rom 6 hours to~less ~ ,, , than;t~o ~.ours.~ The new~method does not requlre as ` ~ intense heating as~the prior art method.

~ 8Lt ~
i~ WO941~gX PC~/VS93/07329 1`` ``

-1 1 - ~ . .`' . The neutral polyglucose used in the present ! :
method for preparing the soluble phosphorylated glucan , ~;
can ke particulate glucan isolated from the cell wall~ , "
of S. cerevisiae by known methods (see e.g., DiLuzio et al., 1979, Int'l J. Cancer 224:773-779; Hassid et al., 1941, J. Amer. Chem. Soc., 63:295-2g8).
Additionally, soluble phosphorylated glucan can be ;`
prepared from neutral polyglucose or polyglucose-protein products deri~ed from a variety of microbial sources. A non-exhaustive list of such sources is presented in Table } of U.S. Patent No. 4,~39,046 incorporated herein by re~erence.

5. 2 SOLUE~LE GLUCAN PRODUCTS AND THEIR_SES
The aqueous soluble glucan products prepared ~i~
according to the method of the present invention are non-toxic, non-pyrogenic and non-immunogenic when evaluated using the interf~acial ring test. As i demonstrated in Exa~ple 7 infra, the soluble phosphor~lated gIucan prepared using the~ impro~ed method of the present invention is substantially the same in composition as that formed by the prLor art method. ~`
The soluble glucans prepared using the ii 25 present method exert profound immunobiological `:
~ responses when a~ministered in vivo. More ^~
`~ particularly, because they are active 45; biological~
response modifiers, the products obtained using the ~relsent method~are useful for prophylaxis and therapy of infectious diseases induced by a variety of microorganisms including, bu~ not llmited to, bacteria, virus, fungi and protozoal parasites.
A~ditionally, the soluble glucans may be used for t~.e l;
prevention and/or~treatment of opportunlstic infections in anlmals~and man which are~

~: , W094/0~98 ~1 4~'1;j PCT/US93/07329~`

immunosuppressed as a result of congenital or a~uired immunodeficiency. -Due to the inability to stimulate macrophage activity and proliferation, the soluble glucans can be 5 used, alone or in combination for therapy of neoplasms.
Routes of administration include, but are not limited ~o: oral, injection, including but not limited to intravenous, intraperitoneal, subcutaneous, l0 intramuscular, and topical routes. The soIuble glucans can be administered in combination with water, an aqueous solution or any~physiologically acceptable ~;
carrier. r ~ ~' The following series of examples are l5 presented for purposes of illustration and not by way of limitation on the scope of the invention.
, 6. PREPARATION OF SOLUBL2 PHOSPHORYLATED GLUCAN
Particulate glucan was prepared~from 20 SaccharomYces cerevisiae according to the method of DiLuzio et al., 1979, Int'l J. Cancer 24:773-779.
Briefly, using a 6 1 flask, 540 ~m of dry yeast - `
(Universal Foods Corp., Milwaukee, WI) was suspended : in 3% aqueous sodium hydroxide solution to a total 25 volum~ of 5 l. The suspension was placed in boiling water bath for 4 hours, cooled overnight and the supernatant decanted.~ This procedure was repeated three times. The residue was then acidified with 5175% hydrochloric acid to a total volume of~5 l~and ~ 3 30 placed in a boiling water bath for 4 hours. The suspension was allowed to stand overnight and the supernatant decanted. The residue~was further , digested twice with of 3% hydre~hlor1c acid to a total ~`~ volume of 5 l at 100C. The residue was~washed with 35 boiling water and decanted numerous times until the~

~:
.

2 1 ~ 1 8 ~
`~`` W0~4/0349~ PCT/US~3/~7329 1``" ```
-13- ~
'~' `.`
residue became floceulent. One 1 of ethyl alcohol was added to the residue, mixed thoroughly and allowed to stand a minimum of 24 hours for maximum extraction. ` " ;~
The dark reddish-brown alcohol supernatant was aspirated from the residue and discarded. The alcohol extraction procedure was repeated until the alcohol superrlatant was essentially colorless. The alcohol was removed by washing the residue four times with hot water; the particulate glucan preparation~was then collected by centrifugatian, ~rozen and~lyophilized.
Soluble phosphorylated glucan was prepared ;~
according to the present invention by solubilization and phosphorylation of the particulate glucan as `~
follows~
18 gm of urea was mixed with l gm of`
particul~te glucan and ground with a pestle in a mortar to form a finely ground powder mixture.
Twenty-fivs ml of phosphoric acid ~43%) was added slowly to the powder mixture to form a slurry. The 20 mixture was heated to about 60-80C and maintained at ~, that temperat.ure for about 1-2 hours with stirring. A ` `;`
precipitate was formed which became visib~e after ~`
about 1-1.5 hours and increased in amounts thereafter. `
About 10 ml of distilled water ~Milli-Q water) was ~`
added to the mixture and heating continued with stirring. The addition of about 10 ml of distilled water was repeated three times and~heating continued j i~
for 1~2 hours. The mixture was then removed from the heat, cooled and diluted with a~out 1 l of distilled 30 water to resuspend the precipitate. The~mixture was ' filtered using a series of filters~to rémove any remaining precipitate. ;~
The resulting solution contalning ~he - ~
soluble phosphorylated glucan was then molecularly ~ ;
sieved to remove low molecular weight ;fractions, , .

~i .
2 1 ~ 1 8 ll 3 PCT/USg3/07329~

including glucose and urea. In one~series of experiments, the mixture~was filtered through coarse (1-3 ~), medium (0.R ~, Q.65 ~) and fine (0.45 ~) sintered Millipore filters to remove the precipitate.
5 The solution was then molecularly sieved using a `;
Millipore dialyzer/concentrator (Millipore Corp., Bedford, MA) with a 10,000 MW membrane filter.
Dialysis against about 24-l00 L of distllled water (Milli-Q grade water) was used to remove~low MW
compounds~
Following molecular sieving, the solution :
containing ~he soluble~phosphorylate glucan was ~ ~
concentrated and lyophilized.~ This yleld was about "
9?%.

7. CHARACTERI~ATION OF SOLUBLE
PHOSPHORYLATED GLUCAN
; PREPA~ED ~Y THE PRESENT METHOD
` A series of~ experiments were conducted to `~compare the soluble~phosphorylated glucan (designated `~
Glucan Phosphate-no DNSO)~prepared;using~the method of the invention as described in Section 5~above with the solùble phosphorylated~qlucan (designated Glucan Phosphate) prepared~using the~prior;~art;method, i.e.,~
the method of U.~S. Patent No. 4,739,~046.

~ ~ .
7.1;~ELEMENTAL COMPOSITION ;:~
The elemental composition~of~the;~Glucan~
Phosphate-no DMSO~was determined by Galbraith Laboratories,~Knoxville, TN. ~A comparLson~of;the elementa} composition~wlth ~that of~Glucan Phosphate~is~
shuvn in ~able l.

2141~
; ~` ` WO 94~03498 " ~Cr!US93/07329 --1 5~

,,, Table 1 , ~ ~
Chemical Composition oî Soluble Glucans - - - _ -Glucan : Glucan Phosphate Phosphate . Mole % (No DMSC" . .
Element Mole % ~:
_ _ _ _ ~

Carbon 34.66 ~ 32.72 Hydrogen 6.~9 : 6.3~
Oxygen 42.83 ~ 48.67 `
Phosphorus 2.23 4 .37 ;.
_ . _ .~...... ,,~

The elemental composition of Glucan Phosphate prepared according to the method of the `,'~"
present invention indicates that it is essentially identical to the,art Glucan Phosphate. As shown in ' '.'' i Table 1, the only difference seen between the product ~'.' of the present method i.e. Glucan Ph~sphate-no DMSO
and the art Glucan Phosphate is the degree of phosphorylation. Glucan Phosphate-no DMSO has ;i ', approximately one phosphate substitution for e~ery 3 ,',;
: glucose units, whereas Glucan Phosphate has 1,,,"
approximately one phosphate substitution for every 7 ; "
glui~ose units. . ,~

7.2 MOLECULAR WEIGHT DISTRIBUTION : ~ ';
The molecular weight (polymer)~dist'ribution!
of the two glucans was determined by aqueous gel permeation chromato~raphy ~GPC). The basic GPC system consisted of a Waters 600E solvent dellvery system, a ~' U6K manual injector 'and a column heating chamber '' ~(Waters Chromatography Divis~ion,~Milllpore Corp., .~ ', Milford, MA). The mobile phase:, 0~.05 M sodium ,~

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W094/0~98 PCr!US93~0732g``: ~.

nitri~e, wàs stored in a sterile reservoir (Kontes, ;-Vineland NJ), and was thoroughly degassed by sparging `
and blanketing with helium prior to use. Mobile phase was delivered at a flow rate of 0.5 ml/min. Three Ultrahydrogel (Waters Chromatography Division, Mi~ford, MA) aqueous GPC columns having excluslon limits of 2 x lo6 D~ 5 x lOs D and 1.2 x 105 D were ~
connected in serles along with~an Ultrahydrogel~guard ;
column. The columns were~malntained at~3~C~. Flow -rate, column temperature, and pump oper~ting~
conditions were controlled by Maxima 8~2a~Gpc software (Dynamic Solutions, Ventura CA).
The system was calibrated using narrow-band ~-pullulan standar~s~and~dextran standàrds. ;For~
15~ analysis, the glucans were~diss~olved in~mobile~phase at a~concentration;of~2-3 mg/ml by gentle~rocking unt~ completely~hydrated~(about 2-3~hrs). A 200 ~l injection volume~was~used for alI~anaLyses.~
Absolute molecular weights of the glucan were determined by on-~line multi-angle~ laser light scattering (MALLS) photometry employing à~Dawn-F MALIS
phot~ometer fitted with~a~K5 flow~cell~(Wyatt; ~
;~ Technology Corp., Santa~Barbara,~CA);.~ Absolute~MW
` distrLbution, molecu;lar~weight mome~nts (number-average ~MW,~Z-average MW~ weight-average~MW),~peak~MW, polyd~ispersity and~root~ mean square~(rms`)~radius ;~ moments were established~with ASTRA software ~v. 2.0).
A differentia} index~of refraction (dn/dc) of 0.146 cm3lg~was a~ssumed~ Reported~MWs of~pullulan and ~
dextran standards~used~to~;check column~calibrat~lon showed g~od agreement~with MALLS~data.~
Intrins~ic~viscosity ([~]~ of~the polymerlc glucans~were~ termined~by~on~ ne~differentlal viscometry ~d;.v.~ For determinat~ion~of~ ] the 35~ Folu eluent was passed~-hrougb~a~1lscoter Modol 200 ````~' W094/03498 21~3 ~5 PCT/U593/073~9 ~ --17- ! ``~
., ` I `'-``~
differential refractometer/viscometer and data were analyzed with Unical software (Viscotek, Porter, TX).
Molecular weight determinations of standards using this technique showed good agreement with MALLS data. ; .
5 Intrinsic viscosity of pullulan standards was ! `~`
determined to be in c~ose agreement with previous . ~-:
data. The molecular weight averages,:polydispersity, .
and intrinsic viscosity of glucan phosphate without `
DMS0 are shown in Table 2. For comparison, analogous ~;
data for glucan phosphate prepared according to the method of Patent No. 4,739,046 are also:presented.

r Wei~ht Characteristics .
_ - _ Para- Glucan Phosphate Glucan Phosphate meter ~ ~ . ¦ no D SO_ _ j ~ peak 1 j peak 2 I peak 1 _ peak 2 `, M~, 1.28 x 1060.25 x 105 2.7~ x 106 0.18 x 105 Mw 3~57 x 1061.10 x 105i 5.45 x 10 1.10 x 105 . ,~

ML 1.22 x 10~3.04; x 105 1.47 x 107 2.75 x 10S `,.
¦ (nm) ¦ 1 25-4 ¦ 40 3 ¦ b Poly- 3.2 6.2 1;.~7 6~1 i dispers .-ity ~I) : :
1 ~1] I C 0.29 ~ c ~ 0.30 ___ a Mn represents: number-average MW s M~" represents: weight-average MW ~ .;Mz representsi: z-average MW : : ~
Mw RMS Radius: weight-average root-me~n square radius (nm? ,L ,' All MW are expressed as g/mol. ~ represents Intrinsic Viscoslty b The RMS Radius cou~d not be determined for this: portion of the sample. c.
c Due to the low concentration of poiymers: in~ peak l ,~ it wasi not possible: ;
35 to determine intrinsic visc05ity io this ponion~of the sample.

: ~:

21~18~ ~
W0~4/0349~ PCT/US93~07329 ; ` `

As demonstrated in Table 2, the molecular weight characteristics of glucan phosphate-no DMSO and the prior art glucan phosphate are strikingly similar, in fact, substantially iden~ical. Using the presently described techniques, two peaks were noted in each of the glucan phosphate praparaticins. The greater major ty of the polymers of both preparations, however were found in peak #2; in both preparations, peak #1 comprises S 2% of the total pol~mers. As clearly ~`~
shown in Table 2, ~, indicative of the~proportion of low MW polymers, Mw, indicative of the average molecular weight, and ~, indicative of the proportion ~``;
of high MW polymers were substantially identical for peak #2 in the two glucan phosphate preparations.
Additionally, the index of polydispersity was substant ally identical showing that compositions had identical polymer homogeneity. Final~y, as shown in Table 2, t~e intrinsic viscosity of the two compositions was virtually identical.
7.3 CONFORMATIONAL STRUCTURE ANALYSIS
Conformational structure was assessed using ~;
the technique of Ogawa and co-workers (Ogawa and Hatana, 1978, Carbohyd. Res. 67:527-53~5; Ogawa and Tsurugi, 1973, Carbohyd. Res. 29:397-403). This ~
~echnique determines the absorption maxima of polymer . ~:
solutions complexed with~Congo Red in the presence of various concentrations~of hydroxide ion. The presence ; j lofj a txipl~e-heli~al compound, for~ example~,~ would,be indicated by a shi~t in the absorption maxima of the solution as sodium hydroxide concentration increases. ~ ~ -Disruption of hydrogen bonds occurs~with the ~ j ~` relaxation of the poIymer helix and subseque.ltly, the Congo Red complexes~with the carbohydrate.

:: ~
~ ; ~ . . ...

2 1~ 4~
~ WOg4/03498 PCT~US93/07329 i -"'~' ., .~
~ It has been previously shown that an ordered (e.g. helical) conformation is essential for i carbohydrates such as glucan to form complexes with I `
the d~e Congo Red. Aqueous solutions of congo Red (44 ~M) were prepared at various concentrations o~ NaOH (1 mM to 1000 mM). Results are presented in FIG. 1 (A
and B) for Glucan Phosphate-no-DMSO and Glucan Phosphate, respectively.
As shown in FIG. 1 ~A and B),~analysis of lO the conformational structure of both the Glucan '`''?"~`' Phosphate and Glucan Phosphate-no DMSO indicates an ordered or ~riple-helical conformation. In both cases, a shift in the absorption maxi~a àt sodium ;
hydroxide concentration of~O.1 to about 0.4 M were observed.
~;
, ~
7 . 4 NUCLEAR RESONANCE SPECTROSCOPY ~;
Carbon-13 nuclear magnetic resonance ,' spectroscopy (13C-NMR) using a Bruker 260 MHz NMR ;`
~0 spectrometer (Bru~er Instruments, Inc., Billerica, MA) ;;~
was performed to determine the nature~of interchain linkages in Glucan Phosphate and Glucan Phosphate-no- i~
DMSO. Soluble Glucan Phosphate or Glucan~Phosphate-no ~i DMSO was dissolved in D20 at 50 mgjml.~; Conditions were ; ~`
as follows~
. "~
FIELD STRENGTH: 50 MH;
RELAXATION DELAY: 1 second, PULSE WINDOW: 15-20, , ;~
NUMBER OF SCANS: Glucan phosphate 694 scans Glucan phosphate-no~DNS0,~1;4~,900 scans.

: , ,! ~
~ Resul~s are showii in FIG.~2 (A-B) and Table 3.
, , ~: .

: ::

21~1Sll~ Pcr/~s93/o73 -20~

T~?le: 3 '~ Chemical Shifts o~ Glucan~* ~ .
__ . _ ~ ! " ` ,~
s ~Glucan Phosphate : :`
Carbon Atom Glucan Phosphate~No DMSO) , . _ _ _ _ C-li 102.58 105.02 ~:"
C-2 73.~3 75.88 C-3 84.45 86.76 ~o C-4~ 68.21 70.7g C ~b 70. 37 C 5 75.66 78 26 C-6 ~0.81 63.40 ~
I .. ,. ._ . _ '. `
Chemical Shifts in ppm ;:~
The only difference between Glucan Phosphate `
and Glucan Phosphate-no DMSO is that Glucan Phosphate-no DMSO shows peaks C4b~and csb, which do not appear in ~i Glucan Phosphate. It is apparent, however, that the compo~nds show IlC-NMR spectra which agree well with a ~1,3-linkage (Colson, Carbohydrate Research 71:265, 2S 1979)-The invention described and~claimed herein ' i5 not to be limited in scope by the specific ~ :
ombodiments herein disclosed, since these embodiments ~ :
are intended as illustrations~of seyeral laspects of , the invention. Any equivalent embodiments are intended to be within;the scope of thls lnventlon.
Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. ~Such modifLcations are also ~ "

21~1~4.~
~`" W094/03498 P~T/US93lO7329~
. -ZI- `";

intended to fall within the scope of the appended ~::
claims. ~ number of re~`erences are cited, the disclosure of each of which is incorporated herein in its entirety by reference.
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Claims (7)

WHAT IS CLAIMED IS:

1. A method for preparing a soluble phosphorylated glucan, comprising:
(a) mixing a neutral polyglucose or a polyglucose-protein complex with a strong chaotropic reagent and grinding the mixture to form a fine powder;
(b) reacting the fine powder with a strong solution of concentrated phosphoric acid to form a reaction mixture containing soluble phosphorylated glucan; and (c) recovering the resultant soluble phosphorylated glucan from the reaction mixture.

2. The method according to claim 1 in which the chaotropic reagent comprises urea.

3. The method according to claim 1, which further comprises heating the reaction mixture of step (b) to about 60-80°C for about 1-6 hours.

4. The method according to claim 3, in which the reaction mixture is heated to about 60-80°C:
for about 1-2 hours.

5. The method according to claim 1, in which the soluble phosphorylated glucan can is recovered by:
(a) allowing the soluble phosphorylated glucan to precipitate;
(b) adding a sufficient amount of water to resuspend the precipitated soluble.
phosphorylated glucan; and c) removing all components of less than about 10,000 daltons molecular weight.

6. The method according to claim 1, in which the neutral polyglucose comprises particulate glucan obtained from a microbial organism.

7. The method according o claim 6, in which the particulate glucan was obtained from Saccharomyces cerevisiae.