Note: Descriptions are shown in the official language in which they were submitted.
2 ~ 42a > >
NOVF;L GLUCAN PREPARATION
Backc~-ound of the Inv~e ' n
'In the early 196~0's, zyi~osan, a crude insoluble yeast
extract prepared by boiling yeast before and after trypsin
treatment, was noted to produce marked hyperplasia and
functional stimulation of the reticuloendothelial system
in rodents. In animal studies, zymosan preparations were
shown to inactivate complement component C3, to enhance
antibody formation, t:o promote survival following
irradiation, to increase resistance to bacterial
infections, to inhibit tumor development, to promote graft
rejection, and to inhibit dietary-induced
hypercholesterolemia and cholesterosis. Zymosan was shown
to consist of polysaccharides, proteins, fats, and
inorganic elements; however, subsequent studies identified
the active components. of the yeast cell wall as a pure
polysaccharide, specifically ~-glucan. In conventional
nomenclature, the po7.ysaccharide ~-glucan is known as
poly-(1-6)-~-D-glucopyranosyl-(1-3)-~-D-glucopyranose
(PGG). Repetition oi: biological assays with ~-glucan
indicated that most of the above functional activities
identified With zymo=:an were retained by the purified ~-
glucan preparation.
The properties of ~-glucan are quite similar to those
of endotoxin in. incrE:asing nonspecific immunity and resis-
tance to infection. The activities of ~-glucan as an
immune adjuvant. and hemopoietic stimulator compare to
those of more complex biological response modifiers
(BRMs), such as. baci7Llus Calmette-Guerin (BCG) and
Corynebacteriua~ parvum. The functional activities of
yeast /~-glucan are a7Lso comparable to those structurally
similar carbohydrate polymers isolated from fungi and
plants. These higher- molecular weight (1-3)-(3-D-glucans
such as schizoF;hyllan, lentinan, krestin, grifolan, and
s
WO 94/04163 ~ ~ ~ ~ PCT/US93/0791?~"
-2-
pachyman exhibit similar immunomodulatory activities. A
common mechanism shared by all these ~-glucan preparations
is their stimulation of cytokines such as interleukin-1
(IL-1) and tuumor nE:crosis factor (TNF) . Lentinan has been
extensively investigated for its antitumor properties,
both in animal modE:ls at 1 mg/kg for 10 days and in
clinical trials since the late 1970s in Japan for advanced
or recurrent malignant lymphoma and colorectal, mammary,
lung and gastric cancers. In cancer chemotherapy,
lentinan has been administered at 0.5-5 mg/day,
intramuscularly (I.M.) or intravenously (I.V.), two or
three times F~er week alone, or in combination with
antineoplasti.c drugs. In addition to the activities
ascribed to ~~east glucans, studies suggest lentinan acts
as a T-cell immunopotentiator, inducing cytotoxic
activities, including production of interleukins 1 and 3
and colony-st:imulaiting factors (CSF). (Chihara gt
1989, IntJ ~J ~~hnmu~igtherativ, 4_:145-154 ; Hamuro and
Chihara, In j~entinan, An Immuno~otentiator)
Various preparations of both particulate and soluble
~-glucans have been tested in animal models to evaluate
biological acaivities. The use of soluble and insoluble
S-glucans alone or as vaccine adjuvants for viral and
bacterial ani:igens has been shown in animal models to
markedly increase :resistance to a variety of bacterial,
fungal, protozoan .and viral infections. The hemopoietic
effects of ~~-glucan have been correlated with increased
peripheral blood leukocyte counts and bone marrow and
splenic cellularity, reflecting increased numbers of
granulocyte-~aacrophage progenitor cells, splenic
pluripotent stem cells, and erythroid progenitor cells, as
well as, increased serum levels of granulocyte-monocyte
colony-stimulating factor (GM-CSF). Furthermore, the
zl4zs~~
--~WO 94/04163 PCT/US93/07904
-3-
hemopoietic an~3 anti-infective effects of ~-glucan were
active in cyclnphosphamide-treated immunosuppressed
animals. S-glvucan was shown to be beneficial in animal
models of trauma, wound healing and tumorigenesis.
However, various insoluble and soluble preparations of ~-
glucan differe~3 significantly in biological specificity
and potency, with effective dosages varying from 25 to 500
mg/kg intravenously or intraperitoneally (I. P.) in models
for protection against infection and for hemopoiesis.
Insoluble preparations demonstrated undesirable
toxicological properties manifested by hepatosplenomegaly
and granuloma formation. Clinical interest was focused on
a soluble glucan preparation which would retain biological
activity yet yield negligible toxicity when administered
systemically. Chronic systemic administration of a
soluble phosphorylated glucan over a wide range of doses
(40-1000 mg/kg) yielded negligible toxicity in animals
, X4_:773-779;
(DiLuzio g~ ~., 1979, Int. J. of Cancer
DiLuzio, U.S. Patent 4,739,046).
The molecular mechanism of action of ~-glucan has
been elucidated by the demonstration of specific ~-glucan
receptor binding sites on the cell membranes of human
neutrophils and macrophages. Mannans, galactans, a(1-4)-
linked glucose polymers and ~(1-4)-linked glucose polymers
have no avidity for this receptor. These ~-glucan binding
sites are opsanin-independent phagocytic receptors for
particulate activators of the alternate complement
pathway, similar to Escherichia coli lipopolysaccharide
(LPS) and some animal red blood cells. Ligand binding to
the ~-glucan receptor, in the absence of antibody, results
in complement activation, phagocytosis, lysosomal enzyme
release, and F~rostaglandin, thromboxane and leukotriene
generation; thereby increasing nonspecific resistance to
WO 94/04163 ~ ~ ~ ~ ~ ~ PCT/US93/0790~
-4-
infection. However, soluble ~-glucan preparations
described in the prior art demonstrated stimulation of
cytokines. Increases in plasma and splenic levels of
interleukins 1 and 2 (IL-1, IL-2) in addition to TNF were
observed 'fin 'vo and corresponded to induction of the
synthesis of these cytokines 'fir vitro. (See Sherwood gt
1987, Int. J. ImmunoDharmac., x:261-267 (enhancement
of IL-1 and IL-2 levels in rats injected with soluble
glucan); Williams et ~., 1988, Int. J. Immunopharmac.,
x:405-414 (systemic administration of soluble glucan to
AIDS patients increased IL-1 and IL-2 levels which were
accompanied by chills and fever); Browder et ~1., 1990,
Ann. Sura., x:605-613 (glucan administration to trauma
patients increased serum IL-1 levels, but not TNF levels);
Adachi ~t ~1_. , 1990, Chem. Pharm. Bull . , ~: 988-992
(chemically cross-linked ~(1-3) glucans induced IL-1
production in mice).)
Interleukin-1 is a primary immunologic mediator
involved in cellular defense mechanisms. Numerous studies
have been carried out on the application of IL-1 to
enhance non-specific resistance to infection in a variety
of clinical states. Pomposelli ~t ~., J. Parent. Ent.
Nutr. 12j2):212-218, (1988). The major problem associated
with the excessive stimulation or exogenous administration
of IL-1 and other cellular mediators in humans is toxicity
and side effects resulting from the disruption of the
gentle balance of the immunoregulatory network. Fauci et
Ann. Int. Med., X06:421-433 (1987). IL-1 is an
inflammatory cytokine that has been shown to adversely
affect a variety of tissues and organs. For instance,
recombinant IL-1 has been shown to cause death,
hypotensive shock, leukopenia, thrombocytopenia, anemia
and lactic acidosis. In addition, IL-1 induces sodium
214211
-5-
excretion, anorexia, slow wave sleep, bone resorption,
decreased pain threshold and expression of many
inflammatory-associated cytokines. It is also toxic to
the insulin secre=ting :beta cells of the pancreas.
Patients suffering from a number of inflammatory diseases
have elevated le~~els of IL-1 in their systems.
Administration o:E agents that enhance further IL-1
production only exacerbate these inflammatory conditions.
Tumor necroais factor is also involved in infection,
inflammation and cancer. Small amounts of TNF release
growth factors w',hile in larger amounts, TNF can cause
septic shock, aches, pains, fever, clotting of blood,
degradation of bone and stimulation of white blood cells
and other immune defenses.
Summary of the Invention
The present invention relates to neutral soluble ~-
glucans which enhance a host's immune defense mechanisms
to infection but do not induce an inflammatory response, -
to preparations containing the neutral soluble ~-glucans,
and to a novel m.anufac;turing process therefor and to their
use in manufacturing medications and in their use in
pharmaceutical c:ompos:itions. In the
present method, solub7.e glucan Which induces cytokine
production is pr'ocesse'd through a unique series of acid,
alkaline and neutral treatment steps to yield a conforma-
tionally pure neutral soluble glucan preparation with
unique biological properties. The neutral soluble glucan
preparation retains a specific subset of immunological
properties common to /3-glucans but uniquely does not
induce the production of IL-1 and TNF in vitro or 'fin vivo.
Throughout this speci:Eication, unless otherwise indicated,
the expressions "neut:ral soluble glucan" and "neutral
soluble ~-glucan" refer to the composition prepared as
described in Example 1.
WO 94/04163 PCT/US93/07904 ._
21428'1 i
-6-
The neutral soluble glucan preparation is produced by
treating insohible g:lucan with acid to produce a water
soluble glucan,, dissociating the native conformations of
the soluble glucan at alkaline pH, purifying the desired
molecular weight fraction at alkaline pH, re-annealing the
dissociated glucan fraction under controlled conditions of
time, temperature and pH to form a unique triple helical
conformation, rind further purifying under neutral pH to
remove single helix .and aggregated materials to yield a
conformational:ly pure, neutral, water soluble,
underivatized c;lucan which has a unique biological
profile .
The neutral soluble glucan preparation has a high
affinity for the ~-glucan receptor of human monocytes and
retains two primary biological activities, (1) the
enhancement of microbicidal activity of phagocytic cells,
and (2) monocyte, neutrophil and platelet hemopoietic
activity. Unlike soluble glucans described in the prior
art, the neutral soluble glucan of this invention neither
induces nor primes mononuclear cells to increase IL-1 and
TNF production ,~ y~t~o and 'fin vivo.
The neutral soluble glucan preparation is appropriate
for parenteral (e. g., intravenous, intraperitoneal,
subcutaneous, intramuscular), topical, oral or intranasal
administration to humans and animals as an anti-infective
to combat infection associated with burns, surgery,
chemotherapy, bone marrow disorders and other conditions
in which the ivmmune system may be compromised. Neutral
soluble glucan produced by the present method can be
maintained in a clear solution and equilibrated in a
pharmaceutically acceptable carrier. Safe and efficacious
preparations of the neutral soluble glucan of the present
invention can be used in therapeutic and/or prophylactic
2142811
_,_
treatment regimens of humans and animals to enhance their
immune response,, without stimulating the production of .
certain biochemical mediators (e.g., IL-1 and TNF) that .
can cause detrimental side effects, such as fever and
inflammation.
Brief Description of 'the Figures
Fig. 1 shoi~s the general structure of neutral soluble
glucan as being a linear /3(1-3)-linked glucose polymer
having periodic branclxing via a single ~(1-6)-linked
to glucose moiety.
Fig. 2 shows a gel permeation chromatogram (pH 7) of
soluble glucan i'hich laas not been purified by alkali
dissociation an~i re-annealing. The chromatogram shows
three species, referred to herein as high molecular weight
aggregate (Ag), Peak i~ and Peak B (single helix glucan).
Fig. 3 is a chromatogram obtained for the neutral
soluble glucan by gel permeation chromatography. The
solid line.reprEaents the neutral soluble glucan at pH 7
and the broken line represents the neutral soluble glucan
at pH 13.
Fig. 4 is a chromatogram obtained for the single
helix ~-glucan y;Peak 33) by gel permeation chromatography.
The solid line represents Peak B at pH 7 and the broken
line represents Peak 13 at pH 13.
Fig. 5 sho~rs the change in serum TNF levels, over
time, taken frolll patients intravenously infused with
placebo (broken line) or neutral soluble glucan (solid
line) .
Fig. 6 shoc~~s the change in serum IL-1 levels, over
time, taken from patients intravenously infused zaith
placebo (broken line) or neutral soluble glucan (solid
line) .
iB
WO 94/04163 PCT/US93/07904 .,~
214211
_8-
Fig. 7 is a diagram representing peripheral blood
counts from irradiated mice following administration of
neutral soluble glucan.
Fig. 8 is a diagram representing platelet cell counts
from cisplatin-treated mice following administration of
neutral soluble gluca:n.
Detailed Descrig~g~ of Invention
The invention relates to a neutral soluble ~-glucan
polymer that ca.n bind to the ~-glucan receptor and
activate only a. desired subset of immune responses. The
terms "neutral solub7.e ~-glucan" and "neutral soluble
glucan", unless, otherwise specified, refer to the
composition prepared as described in Example 1.
This neutral soluble ~-glucan has been shown to
Increase the number of neutrophils and monocytes as well
as their direct: infecaion fighting activity (phagocytosis
and microbial x:illinc~). However, the neutral soluble
glucan does not: stimulate the production of biochemical
mediators, such as Ih-1 and TNF, that can cause detrimen-
tal side effecta such as high fever, inflammation, wasting
disease and organ failure. These advantageous properties
make neutral soluble glucan preparations of this invention
useful in the preveni~ion and treatment of infection
because they sE:lectively activate only those components of
the immune system re:aponsible for the initial response to
infection, without sltimulating the release of certain
biochemical mediator:a that can cause adverse side effects.
The solution containing the neutral soluble ~-glucan also
lacks the toxicity common to many immunomodulators.
The neutral soluble ~B-glucans of this invention are
composed of glucose monomers organized as a ~B(1-3) linked
glucopyranose backbone with periodic branching via ~(1-6)
'WO 94/04163 214 2 ~ 1 i PCT/US93/07904
-g-
glycosidic linkages. The neutral soluble glucan prepara-
tions contain glucans;, which have not been substantially
modified by sub~stitut:ion with functional (e. g., charged)
groups or other covalent attachments. The general
structure of the neutral soluble glucan is shown in Fig.
1. The biologically active preparation of this invention
is a conformationally purified form of ~-glucan produced
by dissociating' the native glucan conformations and re-
annealing and purifying the resulting unique triple
helical conformation. The unique conformation of the
neutral soluble: glucan contributes to the glucan's ability
to selectively activate the immune system without
stimulating the: production of detrimental biochemical
mediators.
The neutral soluble glucan preparations of this
invention are prepared from insoluble glucan particles,
preferably derived from yeast organisms. See Manners ~t
~, Biochem. J_, ~i:19-30, (1973) for a general
procedure to make insoluble yeast glucans. Glucan
particles which are particularly useful as starting
materials in the present invention are whole glucan
particles (WGP) described by Jamas et ., in U.S. Patent
Nos. 4,810,646, 4,99:?,540, 5,082,936 and 5,028,703, the
teachings of al.l of which are hereby incorporated herein
by reference. The source of the whole glucan particles
can be the broad spectrum of glucan-containing yeast
organisms whicri contain ~-glucans in their cell walls.
Whole glucan particles obtained from the strains
Saccharomvces r._erevi:>iae R4 (NRRL Y-15903; deposit made in
connection with U.S. Patent No. 4,810,646) and R4 Ad (ATCC
No. 74181) are particularly useful. Other strains of
yeast that can be used include Saccharomyces delbrueckii,
Saccharomyces rosei, Saccharomyces microelligsodes,
214281 1
-10-
Saccharomyces c:ar s E~rgensis,-. Schizosaccharom c~ pombe,
Kluyveromyces J.ac 's" Kl.uyveromyces fraQilis,
Kluyveromyces ~~olyspc~rus, Candida albicans, Candida
cloacae, Candicla trop'ca 's
Candida utilis
~iansenula
,
,
win ei ansenu a -,~'v, ~iansenula henricii, hansenula
americana.
A procedure for extraction of whole glucan particles
is described by' Jama:~ et al. , in U. S. Patent Nos.
4,810,646, 4,99'2,540,. 5,082,936 and 5,028,703. For the
l0 purpose of this; present invention, it is not necessary to
conduct the final organic extraction and wash steps de-
scribed by Jama.s et
In the preaent process, whole glucan particles are
suspended in an acid solution under conditions sufficient
to dissolve the: acid--soluble glucan portion. For most
glucans, an acid solution having a pH of from about 1 to
about 5 and at a temperature of from about 20 to about
100C is sufficient. Preferably, the acid used is an
organic acid capable of dissolving the acid-soluble glue
portion. Acetic acid, at concentrations of from about 0.1
to about 5 M or formic acid at concentrations of from
about 50% to 98% (w/m) are useful for this purpose. The
treatment time may vary from about l0 minutes to about 20
hours depending) on the acid concentration, temperature and
source of whole: glucan particles. For example, modified
glucans having more X3(1-6) branching than naturally-
occurring, or ~~ild-type glucans, require more stringent
conditions, i.e., longer exposure times and higher
' temperatures. This acid-treatment step can be repeated
under similar car variable conditions. One preferred
processing method is described in the exemplification
using glucan derived from S. cerevisiae strain R4 Ad. In
another embodiment oi= the present method, whole glucan
iE
~~!O 94/04163 ~ ~ ~ PCT/US93/07904
-11-
particles from t:he strain, S. cerevisiae R4, which have a
higher level of ~(1-6) branching than naturally-occurring
glucans, are usE:d, and treatment is carried out with 90%
(w/v) formic ac~.d at a0°C for about 20 minutes and then at
85 ° C f or about ?~ 0 minutes .
The insoluble glucan particles are then separated
from the solution by an appropriate separation technique,
for example, by centrifugation or filtration. The pH of
the resulting s7.urry :is adjusted with an alkaline compound
such as sodium hydroxide, to a pH of about 7 to about 14.
The precipitate is co:Llected by centrifugation and is
boiled in purified wai;.er (e.g., USP) for three hours. The
slurry is then resuspeanded in hot alkali having a
concentration sufficient to solubilize the glucan
polymers. Alkaline compounds which can be used in this
step include alts:ali-mental or alkali-earth metal
hydroxides, such as sodium hydroxide or potassium
hydroxide, having a concentration of from about 0.01 to
about 10 N. This step can be conducted at a temperature
of from about 4°'C to about 121°C, preferably from about
20°C to about 100°C. In one embodiment of the process,
the conditions utilized are a 1 M solution of sodium
hydroxide at a temperature of about 80-100°C and a contact
time of approximately 1-2 hours. The resulting mixture
contains solubil.ized c~lucan molecules and particulate
glucan residue amd generally has a dark brown color due to
oxidation of cor.~taminating proteins and sugars. The
particulate residue is removed from the mixture by an
appropriate separation technique, e.g., centrifugation
and/or filtration. In another embodiment of the process
the acid-soluble: glucans are precipitated after the
preceding acid hydrolysis reaction by the addition of
about 1.5 volumea of Eahanol. The mixture is chilled to
WO 94/04163 PCT/US93/07904_
z~~zsm
-12-
about 4°C for two (2) hours and the resulting precipitate
is collected by centrifugation or filtration and washed
with water. The pellet is then resuspended in water, and
stirred for three (3) to twelve (12) hours at a
temperature between about 20°C and 100°C. At this point
the pH is adjusted to approximately 10 to 13 with a base
such as sodium hydroxide.
The resulting solution contains dissociated soluble
glucan molecules. This solution is now purified to remove
traces of insoluble glucan and high molecular weight
soluble glucans which can cause aggregation. This step
can be carried out by an appropriate purification
technique, for example, by ultrafiltration, utilizing
membranes with nominal molecular weight (NMW) levels or
cut-offs in the range of about 1,000 to 100,000 daltons.
It was discovered that in order to prevent gradual
aggregation or precipitation of the glucan polymers the
preferred membrane for this step has a nominal molecular
weight cut-off of about 100,000 daltons. The soluble
glucan is then further purified at alkaline pH to remove
low molecular weight materials. This step can be carried
out by an appropriate purification technique, for example,
by ultrafiltration, utilizing membranes with nominal
molecular weight levels or cut-offs in the range of 1,000
to 30,000 daltons.
The resulting dissociated soluble glucan is re-
annealed under controlled conditions of time (e. g., from
about 10 to about 120 minutes), temperature (e. g., from
about 50 to about 70°C) and pH. The pH of the solution is
adjusted to the range of about 3.5-11 (preferably 6-8)
with an acid, such as hydrochloric acid. The purpose of
this re-annealing step is to cause the soluble glucan to
rearrange from a single helix conformation to a new
2~4~811
VI~n 94/04163 PCT/US93/07904
-13-
ordered triple helical conformation. The re-annealed
glucan solution .is then size fractionated, for example by
using 30,000-70,~D00 NMW and 100,000-500,000 NMW cut-off
membrane ultrafi.lters to selectively remove high and low
molecular weight soluble glucans. Prior to sizing, the
soluble glucans .exist as a mixture of conformations
including random coils, gel matrices or aggregates, triple
helices and single helices. The objective of the sizing
step is to obtain an enriched fraction for the re-annealed
conformation of ;specific molecular weight. The order in
which the ultraf.ilters are used is a matter of preference.
The concent:rated fraction obtained is enriched in the
soluble, biologi~~ally active neutral soluble glucan. The
glucan concentrate is further purified, for example, by
diafiltration using a 10,000 dalton membrane. The
preferred concentration of the soluble glucan after this
step is from about 2 to about 10 mg/ml.
The neutralized solution can then be further
purified, for ex~~mple, by diafiltration, using a
pharmaceutically acceptable medium (e. g., sterile water
for injection, plnosphate-buffered saline (PBS), isotonic
saline, dextrose) suitable for parenteral administration.
The preferred me»brane for this diafiltration step has a
nominal molecular weight cut-off of about 10,000 daltons.
The final concentration of. the glucan solution is adjusted
in the range of .about 0.5 to 10 mg/ml. In accordance with
pharmaceutical manufacturing standards for parenteral
products, the solution can be terminally sterilized by
filtration through a 0.22 ~,m filter. The neutral soluble
glucan preparation obtained by this process is sterile,
non-antigenic, essentially pyrogen-free, and can be stored
at room temperature (e. g., 15-30°C) for extended periods
of time without degradation. This process is unique in
WO 94/04163 PCT/US93/07904 _.
~14~811
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that it results in a neutral aqueous solution of (pH 4.5
to 7.0) immunologically active glucans which is suitable
for parenteral administration.
For purposes of the present invention, the term
"soluble" as used herein to describe glucans obtained by
the present process, means a visually clear solution can
be formed in an aqueous medium such as water, PBS,
isotonic saline, or a dextrose solution having a neutral
pH (e. g., from about pH 5 to about 7.5), at room
temperature (about 20-25°C) and at a concentration of up
to about 10 mg/ml. The term "aqueous medium" refers to
water and water-rich phases, particularly to
pharmaceutically acceptable aqueous liquids, including
PBS, saline and dextrose solutions. The expression
i5 "visually clear" means that at a concentration of 1 mg/ml,
the absorption of the solution at 530 nm is less than OD
0.01 greater than the OD of an otherwise identical
solution lacking the B-glucan component.
The resulting solution is substantially free of
protein contamination, is non-antigenic, non-pyrogenic and
is pharmaceutically acceptable for parenteral
administration to animals and humans. However, if
desired, the soluble glucan can be dried by an appropriate
drying method, such as lyophilization, and stored in dry
form.
The neutral soluble glucans of this invention can be
used as safe, effective, therapeutic and/or prophylactic
agents, either alone or as adjuvants, to enhance the
immune response in humans and animals. Soluble glucans
produced by the present method selectively activate only
those components that are responsible for the initial
response to infection, without stimulating or priming the
immune system to release certain biochemical mediators
~19~~8~~
~~VO 94/04163 PCT/US93/07904
-15-
(e. g., IL-1, TNF, IL-6, IL-8 and GM-CSF) that can cause
adverse side eiFfects. As such, the present soluble glucan
composition can be used to prevent or treat infectious
diseases in ma:lnouri;shed patients, patients undergoing
surgery and bone marrow transplants, patients undergoing
chemotherapy or radiotherapy, neutropenic patients, HIV-
infected patients, trauma patients, burn patients,
patients with chronic or resistant infections such as
those resulting from myelodysplastic syndrome, and the
elderly, all o:E who :may have weakened immune systems. An
immunocompromi,~ed individual is generally defined as a
person who exhibits an attenuated or reduced ability to
mount a normal cellular and/or humoral defense to
challenge by infectious agents, e.g., viruses, bacteria,
fungi and protozoa. A protein malnourished individual is
generally defined as a person who has a serum albumin
level of less 'than about 3.2 grams per deciliter (g/dl)
and/or unintentional weight loss of greater than 10% of
usual body weight.
More particularly, the method of the invention can be
used to therapeutically or prophylactically treat animals
or humans who .are at a heightened risk of infection due to
imminent surgery, injury, illness, radiation or
chemotherapy, or other condition which deleteriously
affects the immune system. The method is useful to treat
patients who have a disease or disorder which causes the
normal metabolic imtfl~une response to be reduced or
depressed, such as HIV infection (AIDS). For example, the
method can be used t.o pre-initiate the metabolic immune
response in patients. who are undergoing chemotherapy or
radiation therapy, or who are at a heightened risk for
developing secondary infections or post-operative
complications because of a disease, disorder or treatment
WO 94/04163 2 ~ 4 2 81 1 PCT/US93/07904
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resulting in a reduced ability to mobilize the body's
normal metabolic responses to infection. Treatment with
the neutral soluble glucans has been shown to be
particularly effective in mobilizing the host's normal
immune defenses, thereby engendering a measure of
protection from infection in the treated host.
The present composition is generally administered to
an animal or a human in an amount sufficient to produce
immune system enhancement. The mode of administration of
the neutral soluble glucan can be oral, enteral, parenter-
al, intravenous, subcutaneous, intraperitoneal,
intramuscular, topical or intranasal. The form in which
the composition will be administered (e. g., powder,
tablet, capsule, solution, emulsion) will depend upon the
route by which it is administered. The quantity of the
composition to be administered will be determined on an
individual basis, and will be based at least in part on
consideration of the severity of infection or injury in
the patient, the patient's condition or overall health,
the patient's weight and the time available before
surgery, chemotherapy or other high-risk treatment. In
general, a single dose will preferably contain
approximately 0.01 to approximately 10 mg of modified
glucan per kilogram of body weight, and preferably from
about 0.1 to 2.5 mg/kg. The dosage for topical
application will depend upon the particular wound to be
treated, the degree of infection and severity of the
wound. A typical dosage for wounds will be from about
0.001 mg/ml to about 2 mg/ml, and preferably from about
0.01 to about 0.5 mg/ml.
In general, the compositions of the present invention
can be administered to an individual periodically as
necessary to stimulate the individual's immune response.
~~O 94/04163 ~ ~ ~ ~ PCT/US93/07904
-17-
An individual sls;filled in the medical arts will be able to
determine the lE:ngth of time during which the composition
is administered and the dosage, depending upon the
physical condition of the patient and the disease or
disorder being treated. As stated above, the composition
may also be used as a preventative treatment to pre-
initiate the normal metabolic defenses which the body
mobilizes against infections.
Neutral soluble /~-glucan can be used for the
prevention and treatment of infections caused by a broad
spectrum of bact:erial,, fungal, viral and protozoan
pathogens. The proph~~lactic administration of neutral
soluble ~-glucar: to a person undergoing surgery, either
preoperatively, intraoperatively and/or post-operatively,
will reduce the incidence and severity of post-operative
infections in both normal and high-risk patients. For
example, in patients undergoing surgical procedures that
are classified as contaminated or potentially contaminated
(e. g., gastrointestinal surgery, hysterectomy, cesarean
section, transurethra:l prostatectomy) and in patients in
whom infection e:t the operative site would present a
serious risk (e.,g., p;rosthetic arthroplasty,
cardiovascular :aurger;Y), concurrent initial therapy with
an appropriate eintibacterial agent and the present neutral
soluble glucan ~~reparation will reduce the incidence and
severity of infE:ctiou;s complications.
In patient:a who are immunosuppressed, not only by
disease (e. g., cancer, AIDS) but by courses of chemother-
apy and/or radiotherapy, the prophylactic administration
of the soluble c~lucan will reduce the incidence of
infections causEad by .a broad spectrum of opportunistic
pathogens including many unusual bacteria, fungi and
viruses. Therapy using neutral soluble ~-glucan has
WO 94/04163 ~ ~ ~ ~ ~ ~ ~ PCT/US93/0790~
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demonstrated a significant radio-protective effect with
its ability to enhance and prolong macrophage function and
regeneration and, as a result enhance resistance to
microbial invasion and infection.
In high risk patients (e. g., over age 65, diabetics,
patients having cancer, malnutrition, renal disease,
emphysema, dehydration, restricted mobility, etc.)
hospitalization frequently is associated with a high
incidence of serious nosocomial infection. Treatment with
neutral soluble ~-glucan may be started empirically before
catheterization, use of respirators, drainage tubes,
intensive care units, prolonged hospitalizations, etc. to
help prevent the infections that are commonly associated
with these procedures. Concurrent therapy with antimi-
crobial agents and the neutral soluble ~B-glucan is
indicated for the treatment of chronic, severe,
refractory, complex and difficult to treat infections.
The compositions administered in the method of the
present invention can optionally include other components,
in addition to the neutral soluble ~-glucan. The other
components that can be included in a particular
composition are determined primarily by the manner in
which the composition is to be administered. For example,
a composition to be administered orally in tablet form can
include, in addition to neutral soluble ~-glucan, a filler
(e. g., lactose), a binder (e. g., carboxymethyl cellulose,
gum arabic, gelatin), an adjuvant, a flavoring agent, a
coloring agent and a coating material (e.g., wax or
plasticizer). A composition to be administered in liquid
form can include neutral soluble ~-glucan and, optionally,
an emulsifying agent, a flavoring agent and/or a coloring
agent. A composition for parenteral administration can be
mixed, dissolved or emulsified in water, sterile saline,
214~~~.1
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PBS, dextrose or other biologically acceptable carrier. A
composition for topical administration can be formulated
into a gel, ointment, lotion, cream or other form in which
the composition is capable of coating the site to be
treated, e.g., 'wound site.
Compositions comprising neutral soluble glucan can
also be administered topically to a wound site to
stimulate and enhance wound healing and repair. Wounds
due to ulcers, .acne, viral infections, fungal infections
or periodontal disease, among others, can be treated
according to the methods of this invention to accelerate
the healing pro~~ess. Alternatively, the neutral soluble
~-glucan can be injected into the wound or afflicted area.
In addition to wound repair, the composition can be used
to treat infection associated therewith or the causative
agents that result in the wound. A composition for
topical administration can be formulated into a gel,
ointment, lotion, cream or other form in which the
composition is capable of coating the site to be treated,
e.g., wound site. The dosage for topical application will
depend upon the particular wound to be treated, the degree
of infection and severity of the wound. A typical dosage
for wounds will be from about 0.01 mg/ml to about 2 mg/ml,
and preferably :from about 0.01 to about 0.5 mg/ml.
Another particular use of the compositions of this
invention is four the treatment of myelodysplastic syndrome
(l~S). MDS, frequently referred to as preleukemia
syndrome, is a ~~roup of clonal hematopoietic stem cell
disorders characterized by abnormal bone marrow
differentiation and maturation leading to peripheral
cytopenia with high probability of eventual leukemic
conversion. Recurrent infection, hemorrhaging and
terminal infection resulting in death typically accompany
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MDS. Thus, in order to reduce the severity of the disease
and the frequency of infection, compositions comprising
modified glucan can be chronically administered to a
patient diagnosed as having MDS according to the methods
of this invention, in order to specifically increase the
infection fighting activity of the patient's white blood
cells. Other bone marrow disorders, such as aplastic
anemia (a condition of quantitatively reduced and
defective hematopoiesis) can be treated to reduce
infection and hemorrhage that are associated with this
disease state.
Neutral soluble glucan produced by the present method
enhances the non-specific defenses of mammalian
mononuclear cells and significantly increases their
ability to respond to an infectious challenge. The unique
property of neutral soluble glucan macrophage activation
is that it does not result in increased body temperatures
(i.e., fever) as has been reported with many non-specific
stimulants of those defenses. This critical advantage of
neutral soluble glucan may lie in the natural profile of
responses it mediates in white blood cells. It has been
shown that the neutral soluble ~B-glucan of the present
invention selectively activates immune responses but does
not directly stimulate or prime cytokine (e.g., IL-1 and
TNF) release from mononuclear cells, thus distinguishing
the present neutral soluble glucan from other glucan
preparations (e.g., lentinan, kresein) and
immunostimulants.
In addition, it has been demonstrated herein that the
neutral soluble glucan preparation of the present
invention possesses an unexpected platelet stimulating
property. Although it was known that glucans have the
ability to stimulate white blood cell hematopoiesis, the
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disclosed platcalet stimulating property had not been
reported or ani~icipa~ted. This property can be exploited
in a therapeutic regimen for use as an adjuvant in
parallel with radiation or chemotherapy treatment.
Radiation and chemotherapy are known to result in
neutropenia (rcaduced polymorphonuclear (PMN) leukocyte
cell count) and thro;mbocytopenia (reduced platelet count).
At present, these conditions are treated Dy the
administration of colony-stimulating factors such as GM-
IO CSF and granulocyte colony-stimulating factor (G-CSF).
Such factors ace effective in overcoming neutropenia, but
fail to impact upon thrombocytopenia. Thus, the platelet
stimulating property of the neutral soluble glucan
preparation of this invention can be used, for example, as
a therapeutic ~~gent to prevent or minimize the development
of thrombocyto~~enia which limits the dose of the radiation
or chemotherap~eutic agent which is used to treat cancer.
The invention is further illustrated by the following
Examples.
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EXAMPLES
EXAMPLE l: PREPARATION OF NEUTRAL SOLUBLE GLUCAN
FROM S. CEREVISIAE
Saccharomvces cerevisiae strain R4 Ad (a non-recombi-
pant derivative of wild-type strain A364A), was grown in a
large-scale fermentation culture using a defined glucose,
ammonium sulfate minimal medium. The production culture
was maintained under glucose limitation in a feed-batch
mode (New Brunswick MPP80). When the growing culture
reached late logarithmic phase, the fermentation was ended
and the ~-glucan was stabilized by adjusting the culture
to pH 12 ~ 0.5 using 10 M NaOH. The yeast cells
containing ~-glucan were harvested by continuous-flow
centrifugation (Westfalia SA-1). After centrifugation,
the cells were collected into a stainless steel extraction
vessel.
The first step in the extraction process was an
alkaline extraction accomplished by mixing the cells with
1 M sodium hydroxide (NaOH) at 90 ~ 5°C for 1 hour. Upon
completion of this alkaline extraction, the ~B-glucan
remained in the sclid phase, which was collected by
continuous centrifugation (Westfalia SA-1). The collected
cell wall fraction was extracted a second time using the
same procedure and under the same conditions. Treatment
with alkali hydrolyzed and solubilized the cellular
proteins, nucleic acids, mannans, soluble glucans and
polar lipids into the supernatant fraction, and deacety-
lated chitin to chitosan in the cell wall.
The second step in the extraction process was a pH
4.5 ~ 0.05 (adjusted with concentrated HC1) extraction at
75 ~ 5°C for 1 hour. This was followed by a 0.1 M acetic
acid extraction to complete the removal of glycogen,
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chitin, chitosan and remaining proteins. The solids were
collected and rinsed itwice with Purified Water USP to
remove any residual acid as well as any yeast degradation
products.
The third step in the extraction process was a set of
six organic ext:-actions. The first four extractions were
carried out in isopropanol. The solids were collected by
centrifugation and then subjected to two acetone extrac-
tions. The two--stage organic extractions eliminated
nonpolar lipids and h!~drophobic proteins which may have
co-purified with the drug substance. The resulting wet
solids were dried in a vacuum oven at 65 ~ 5°C for 48-96
hours to yield ei free~-flowing powder.
At this stage the extraction process yielded a
stable, insolub7le intermediate consisting of approximately
90% ~-glucan, celled tahole glucan particles (WGPs). The
dry WGP intermediate was stored at 15-30°C until further
use.
The WGP powder was resuspended in 98% (w/v) formic
acid, in a glass reacition vessel at room temperature. The
resulting mixture was heated to 85 ~ 5°C for 20 minutes.
Under these condition;a, the WGPs were partially hydrolyzed
and solubilized to provide the desired molecular weight
distribution of soluble ~-glucan which was then
precipitated by adding 1.5 volumes of ethanol. After
complete mixing,, the preparation was centrifuged to
collect the ~-glucan precipitate. Any residual formic
acid was removed by boiling the ~-glucan preparation in
Purified Water Z1SP for three hours.
Any unhydrolyzed WGPs were then removed from the
glucan solution by centrifugation. The ~-glucan solution
was raised to pFi 12.5 ~ 0.5 by the addition of the concen-
WO 94/04163 PCT/US93/0790~-
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trated sodium hydroxide. The remaining purification steps
were carried out by ultrafiltration.
The soluble alkaline ~-glucan preparation was passed
through a 100,000 nominal molecular weight (NMW) cut-off
membrane ultrafilter (Amicon DC10). Under alkaline
conditions this membrane ultrafilter removed insoluble and
high molecular weight soluble ~-glucan. Trace low
molecular weight degradation products were then removed by
recirculation through a 10,000 NMW cut-off membrane
ultrafilter. The ultrafiltration was conducted as a
constant volume wash with 0.1 M NaOH.
The ~-glucan solution was re-annealed under
controlled conditions by adjusting the pH to 7.0 ~ 0.5
with concentrated hydrochloric acid, heating to 60 ~ 10°C,
which was maintained for 20 minutes and then cooled. The
neutral re-annealed solution was then concentrated and
washed with Sodium Chloride Injection USP in a 70,000 NMW
cut-off membrane ultrafilter (Filtron Minisep) to enrich
for the re-annealed neutral soluble glucan. Next the
material was filtered through a 300,000 NMW cut-off
membrane ultrafilter (Filtron Minisep) to remove high
molecular weight and aggregated glucan molecules. In the
same ultrafilter, the neutral soluble glucan material was
washed with Sodium Chloride Injection USP in a constant
volume wash mode.
The neutral soluble glucan was then concentrated in a
10,000 NMW cut-off membrane ultrafilter. The
concentration process continued until a concentration of
at least 1.0 mg/ml hexose equivalent was achieved.
The resulting neutral soluble glucan was then
subjected to filtration through a depyrogenating filter
(0.1 micron Posidyne) and a sterile 0.2 micron filter
(Millipak) to yield sterile, pyrogen-free neutral soluble
"'10 94/04163 2 ~ 't t~ ~ ~ ~ PCT/US93/07904
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glucan. The neutral ~.oluble glucan solution was stored at
controlled room temperature (15-30°C) until further use.
The aqueous solubility of neutral soluble glucan in the pH
range of 4 to 8 is approximately 100 mg/ml. The solubil-
ity increased with increasing pH and reached approx. 150
mg/ml at pH 13.
EXAMPLE 2: ANAhYSIS ()F NEUTRAL SOLUBLE GLUCAN
A. Glucose. Mar~nose rind Glucosamine
Monosaccharide analysis was performed to quantitate
l0 the relative amounts of ~B-glucan (as glucose), mannan or
phosphomannan (ass mannose), and chitin (as N-acetyl
glucosamine) in the neutral soluble glucan. The sample
was hydrolyzed t:o monosaccharides in 2 M trifluoroacetic
acid for 4 hours at 1:L0°C, evaporated to dryness, and
redissolved in water. Monosaccharides were separated on a
Dionex HPLC system using a CarboPac PA100 column (4 x 250
mm) using 5 M Na~OH at 1 ml/min and quantitated using a
pulsed electrocriemica:l detector (Dionex Model PED-1). The
sensitivity of this assay for monosaccharides is 0.1%
(w/w) .
Glucose (reaention time of 16.6 min) was identified
as the only monosaccharide component of neutral soluble
glucan along with traces of glucose degradation products
(from hydrolysi:c) anhl~droglucose at 2.5 min and 5-
hydroxymethylfurfural at 4.3 min. The results confirm
that neutral so7.uble glucan consisted of z98% glucose.
B. FTIR
Fourier tra~nsfona infrared spectroscopy by diffuse
reflectance (FT7:R, Matson Instruments, Polaris) of lyophi-
lined neutral soluble glucan samples was used to determine
the anomeric st:-uctur~e (a vs. ~), and linkage type (~(1-
WO 94/04163 PCT/US93/079Q4
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3), ~(1-6), ~(1-4)) present in neutral soluble glucan.
Absorption maxima of 890 cm'' identified ~(1-3) linkages;
920 cm'' identified ~(1-6) linkages. No presence of c-
linked anomers (e. g., glycogen, 850 cm'') or ~(1-4)-linked
polysaccharides (e. g., chitin, 930 cm') were detected.
EXAMPLE 3: CONFORMATIONAL ANALYSIS
A solution of ~-glucan which was not processed by
alkali dissociation and re-annealing was analyzed for its
compositional identity by gel permeation chromatography
IO (pH 7) and found to contain multiple species, referred to
herein as high molecular weight aggregate (Ag), Peak A and
Peak B (See Figure 2). Neutral soluble glucan which was
prepared by alkali dissociation and re-annealing as
described in Example 1, is present as a single peak (see
Figure 3) with an average molecular weight of 92,660
daltons at pH 7. The distinct conformations of neutral
soluble glucan and Peak B were demonstrated by gel
permeation chromatography at pH 7 and pH 13 using a
refractive index detector. Neutral soluble glucan under-
went a significant conformational transition from pH 7 to
pH I3 which illustrates complete dissociation of the
multiple helix at pH 7 to a single helical form at pH 13
(see Fig. 3). In contrast, Peak B only underwent a slight
shift in molecular weight from pH 7 to pH 13 (see Fig. 4).
The molecular weight of neutral soluble glucan and Peak B
glucans as a function of pH is shown below in Table 1.
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Table 1
Sample MW MW Ratio
pH 7 pH 13 (pH 7/pH 13)
Neutral soluble 92,666 18,693 4.96
glucan
Peak B 8,317 7,168 1.16
.The conformation of neutral soluble glucan and Peak B
glucan was also dete:nained by aniline blue complexing
(Evens gt ~., 1984, Carb. Pol., 4:215-230; Adachi g~ ~.,
1988, Carb. Red., ,x'7:91-100), using curdlan, a linear
~(1-3) glucan, as the triple helix control and pustulan, a
~(1-6) glucan, as a non-ordered conformational control.
The results are discussed below and shown in Table 2.
The curdlan triple helix control complexed with
aniline blue resulting in high fluorescence. Increasing
the NaOH concentration began to dissociate the curdlan
triple helix slightly, but NaOH concentrations >0.25 M are
required for complete dissociation of curdlan. The
pustulan non-ordered control only formed a weak complex
with aniline blue reaulting in low fluorescence
measurements which were not affected by NaOH
concentration.
The neutral soluble glucan complexed effectively with
aniline blue at low NaOH concentration (25mM NaOH)
resulting in high fluorescence. However, the neutral
soluble glucan conformation dissociated significantly
(50$) at NaOH concentrations as low as 150 mM NaOH
WO 94/04163 ~ ~, PCT/US93/07904
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indicating that it exists as a unique conformation
compared to naturally occurring ~-glucans, such as
laminarin and curdlan, which require significantly higher
NaOH concentrations for dissociation to occur. Peak B
formed a weak complex with aniline blue due to its single
helical conformation.
Table 2
Conformational Analysis of Glucans
by Aniline Blue Complexing
Fluorescence
Test Material 25 mM 0~ 0 mM 51 0
mM
NaOH NaOH NaOH
Blank 0 2 0
Curdlan 53.5 41.6 36
~(1-3) glucan
Pustulan 9.8 8.3 8.0
~S ( 1-6 ) glucan
Neutral soluble glucan 40 25.6 20.2
Peak B 12.4 6.2 4.1
EXAMPLE 4: EFFECTS OF NEUTRAL SOLUBLE GLUCAN ON HUMAN
MONOCYTE PRODUCTION OF TNFa
Human peripheral blood mononuclear cells were
isolated (Janusz et ., (1987), J. Immunol., ~: 3897-
5 3901) from normal citrated and dextran-treated blood,
washed in Hank's balanced salt solution (HBSS), lacking
calcium, magnesium, and phenol red, and purified by
gradient centrifugation on cushions of Ficoll-Paque
(Pharmacia Fine Chemicals, Piscataway, NJ). The
mononuclear cells were collected into HBSS, washed twice,
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resuspended in RPMI 1Ei40 Medium (Gibco, Grand Island, NY)
containing 1% heat-inaictivated autologous serum (56°C for
30 min.), and ca~unted on the Coulter counter.
For the preparation of monocyte monolayers, 1 ml of
2.2 x 106 mononuclear cells/ml was plated into wells of
24-well tissue culturE: plates (Costar, Cambridge, MA),
incubated for 1 hour at 37°C in a humidified atmosphere of
5% COZ, and washed three times with RPMI to remove
nonadherent cells. A second 1 ml aliquot of 2.2 x 106
mononuclear cells/ml was layered into each well and
incubated for 2 hours described above before removal of
the nonadherent cells. By visual enumeration at 40X with
an inverted phase microscope and a calibrated reticle, the
number of adherent cells for 30 different donors was 0.77
f 0.20 X 106 per well (mean ~ SD). By morphology and
nonspecific esterase staining, >95% of the adherent cells
were monocytes.
Monocyte monolaye:rs were incubated at 37°C in the COZ
chamber for 0 to 8 hours with 0.5 ml of RPMI, 1% heat-
inactivated autologou:c serum, 10 mM HEPES, and 5 mM MgCh
in the absence and preaence of various glucan prepara-
tions. The culture supernatant was removed, clarified by
centrifugation at 14,000 g for 5 min at 4°C, and stored at
-70°C before assay of TNFa.
The concentration of TNFa in the monocyte super-
natants was measured by an enzyme-linked immunoadsorbent
assay (ELISA) with the: BIOKINE TNF Test kit (T Cell
Sciences, Cambridge, MA), which had a lower limit of
detectability of 40 pc~/ml. The data are expressed as pg
per 106 monocytea, which was calculated by dividing the
quantity of cyto~kine in 0.5 ml of supernatant by the
number of monocytes pe:r well.
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For the determination of cell-associated levels of
TNFa, the adherent monocytes were lysed in 0.25 ml PBS by
three rounds of freezing and thawing, the lysates were
cleared of debris by centrifugation at 14,000 g for 5 min
at 4°C, and the resulting supernatants were stored at
-70°C. Newly prepared monocyte monolayers contained no
detectable levels of intracellular TNFa.
The results are shown in Tables 3 and 4 below.
Table 3
TNFa Synthesis by Human Monocytes Stimulated
with Various Glucan Preparations
TNFa (pg/106 monocytes)
~lucan Conc. 1 2 3 MeanSD
Buffer Control 36 39 2 2621
Neutral soluble img/ml 44 51 33 439
glucan
Laminarin img/ml 372 324 227 30874
Whole
Glucan particles 4X10~/ml 2129 1478 1683 1763333
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Table 4
TNFa Stimulation by Different Conformational
Sl:ructu:res of Soluble ~-Glucan
TNFa
Glucan Conc. (pg/106 monocytes)
Buffer Control 1 mg/ml 40
Laminarin 1 mg/ml 1312
Neutral soluble glucan 1 mg/ml 16
Peak B 1 mg/ml 1341
Glucan Particle:a 4 X 10~/ml 2065
Table 3 shows that TNFa was stimulated by insoluble
glucan particles and by laminarin, a soluble ~(1-6) and
~(1-3) linked g7~.ucan. There was no stimulation of TNFa by
neutral soluble glucan. Table 4 shows similar results,
but further con~:irms that TNFa stimulation is dependent
upon conformational structure. The neutral soluble glucan
did not stimulate TNFa while Peak B (single helical
conformation) did stimulate TNFa.
EXAMPLE 5: ~~VIDIT'i~ OF NEUTRAL SOLUBLE GLUCAN FOR THE
S~LUCAN RECEPTOR
Monolayers of hwaan monocytes, prepared on
siliconized gla:a coverslips (Czop et al., 1978, 7~.
Immunol., ~,~:17.32), were incubated for 18 minutes at 37°C
in a humidified 5% CO, incubator with either 0.25 ml of
buffer (RPMI-Mg--HEPES) or a range of concentrations (0.1-
50 ~g/ml) of neutral :soluble glucan. The monocyte
monolayers were then taashed twice with 50 ml of RPMI 1640
medium and were layered with 0.25 ml of 4.8 x 106/ml
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zymosan particles (Czop and Austere, 1985, J. Immunol.,
x:2588-2593). After a 30 minute incubation at 37°C, the
monolayers were washed three times with 50 ml of Hank's
balanced salt solution to remove noningested zymosan
particles. The monolayers were then fixed and stained
with Giemsa. The ingestion of zymosan particles by at
least 300 monocytes per monolayer was determined by visual
observation under a 1000X light microscope.
Monocyte monolayers pretreated with buffer, 50 or 500
~Cg/ml of neutral soluble glucan as described above were
subsequently tested for their capacity to ingest IgG
coated sheep erythrocytes (E'IgG). After an 18 minute
preincubation with the neutral soluble glucan, the
monolayers were incubated with 0.25 ml of 1 x 10'/ml E'IgG
for 30 minutes at 37°C, washed three times with 50 ml of
Hank's balanced salt solution, treated for 4 minutes with
0.84% NH,C1 to lyse noningested E'IgG, and fixed and
stained as described above. The percentages of monocytes
ingesting >_ 1 and >_ 3 E'IgG were determined by counting at
least 300 monocytes per monolayer.
The percent inhibition of monocyte ingestion was
detenained by subtracting the percentage of monocytes
ingesting targets after pretreatment with the neutral
soluble glucan from the percentage ingesting targets after
pretreatment with buffer, dividing this number by the
percentage ingesting targets after pretreatment with
buffer and multiplying by 100. The data are expressed as
the mean of two experiments and are reported in Table 5.
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Table 5
Glucan-receptor Binding Capacity of
Distinct Conformations of Soluble ~-glucans
Test Material Conc. % Inhibition
Buffer - 0%
Neutral soluble: glucan 50 ~cg/ml 74%
500 ~tg/ml 86%
Peak B 50 ~g/ml 50%
500 ~tg/ml 56%
Both ~-glucan preparations tested above inhibited
monocyte ingestion o~f zymosan particles demonstrating
their capacity to competitively bind to the ~-glucan
receptor on human mo~nocytes. Neutral soluble glucan
demonstrated a higher receptor binding capacity than
Peak B as indicated by the greater level of inhibition
achieved at both 50 ~cg/ml and 500 y~g/ml. This
biological assay demonstrates that the neutral soluble
glucan is a superior ligand for the ~-glucan receptor.
EXAMPLE 6: ~CK,OF IN VITRO STIMULATION OF IL-18
AND Z'NFct FROM HUMAN MONONUCLEAR CELLS
Venous blood ways obtained from healthy male volun-
teers and mononuclear cells were fractionated by
Ficoll-Hypaque centrifugation. The mononuclear cells
were washed, resuspe:nded in endotoxin-free RPMI-1640
culture medium - ult:rafiltered to remove endotoxins as
described elsewhere (Dinarello et al., 1987, J. Clin.
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Microbiol. x:1233-8) - at a concentration of 5 x 106
cells/ml and were aliquoted into 96-well microtiter
plates (Endres stet al., 1989, N.E. J. Med. ~Q:265-271).
The cells were then incubated with either 1 ng/ml
endotoxin (lipopolysaccharide, E. coli 055: B5, Sigma,
St. Louis), or l0 to 1000 ng/ml ~-glucan, at 37°C for
24 hours in 5% COZ and then lysed by three freeze-thaw
cycles (Endres et al., 1989, N.E. J. Med.
~Q:265-271) .
Synthesis of IL-is and TNFa was determined by specific
radioimmunoassays as described elsewhere (Lisi stet al.,
1987, lymph Res. _6:229-244; Lonnemann et ., 1988,
Lvmnh. Res. 7:75-84; Van der Meer et ., 1988, J.
Leukocycte Biol. X3:216-223.
To determine if neutral soluble glucan could act
as a priming agent for cytokine synthesis with
endotoxin, a known cytokine stimulant, mononuclear
cells were pre-incubated with 1, 10, and 1000 ng/ml of
the neutral soluble glucan for 3 hours at 37°C in 5%
C02. The cells were washed to remove neutral soluble
glucan and were then incubated with 1 ng/ml endotoxin
as described above. IL-1~ and TNFa were determined as
described above.
The results are summarized in Table 6. Neutral
soluble glucan used as a stimulant at doses of 10-1000
ng/ml alone did not induce increased levels of IL-1~ or
TNFa synthesis over the control buffer treated cells.
Endotoxin LPS, a known stimulant, resulted in signifi-
cantly increased levels of both cytokines. In a second
phase of this experiment neutral soluble glucan was
tested for its ability to act as a priming agent for
mononuclear cell cytokine synthesis. The cells from
the same donors were pre-incubated with three doses of
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neutral soluble glucan (l0-1000 ng/ml) and were then
exposed to endot:oxin as a co-stimulant. Neutral
soluble glucan did volt result in any amplification of
the IL-1~ and TDfFa le~~els compared to endotoxin alone.
Table 6
In Vitro IL-1~ and TNFa Synthesis by
Human Peripheral Blood Mononuclear Cells
Stimulant IL-1~ TNFa
(ng/ml)' (ng/ml)'
Cells only <0.10 0.14
Neutral 10 ng/ml 0.13 0.16
soluble
glucan x.00 ng,~ml o .12 0.16
1000 ng,~ml <0.10 0.14
LPS 1 ng,~ml 2.62 2.22
LPS(1 ng/ml)+10 ng,~ml 2.62 2.25
Neutral
soluble 1.00 ng,~ml 2.57 2.07
glucan 1000 ng/ml 2.85 2.27
'Values are the mean of two donors.
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EXAMPLE 7: ~N VIVC~ PROTECTION AGAINST _
~NFECTfON IN RATS ,
A sepsis model. was developed in rats to
characterize the efficacy of ~-glucan in protecting an
immunologically intact host against serious infections,
such as those which. commonly occur following abdominal
surgery. The rat model for intra-abdominal sepsis has
been well described in the scientific literature
(Onderdonk et al., 1974, Infect. Immun., X0:1256-1259).
Groups of rats received neutral soluble glucan
(100 ~g/0.2 ml) or saline control (0.2 ml)
intramuscularly 24 hours and 4 hours prior to
infectious challenge. A defined polymicrobic
infectious challenge (cecal inoculum) was placed into a
gelatin capsule which was then surgically implanted
into the peritoneal cavity of anesthetized rats through
an anterior midline incision. The early peritonitis
from this experimentally induced infection was
associated with the presence of gram-negative organisms
within the bl~~od and peritoneal cavity culminating in
mortality. T:he cecal inoculum contained an array of
facultative species, such ~ co ', as well as other
obligate anaerobes (Streptococcus sp., Pacteroides sp.,
Clostridium perfrin ens, Clostridium ramosum,
Peptostreptococcus ~naanus and productus, Proteus mira-
bilis). The animals were observed four times per day
for the first 48h and twice per day thereafter. The
results are reported in Table 7.
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Table ..7
Effect of Neutral So7Luble Glucan on Mortality in a Rat
Model for Intra~-abdominal Sepsis
Group Mortality(%1 P vs. Saline
Saline 12/20 (60)
Neutral soluble glucan 2/10 (10) < 0.01
These results demonstrate that neutral soluble glucan
-- which does not .induce IL-1~ and TNFa protects rats from
lethal bacterial challenge.
EXAMPLE 8: pEMnNSTRA'TION OF SAFETY FOR
SAN ADMINISTRATION
A randomized, double-blind, placebo-controlled
clinical trial was conducted on healthy males to
evaluate the safety of neutral soluble glucan (2.25
mg/kg) injected by intravenous infusion compared to a
placebo control. No adverse effects were observed.
There was also :no observed elevation in IL-1, TNF, IL-
6, IL-8 and GM-~CSF. Single intravenous administration
of neutral soluble glucan resulted in an increase in
mvnocytes and n~eutrophils and in the killing activity
of these cells ;proving that neutral soluble glucan
retains the desirable immunological activities in
humans. See Tables 8, 9 and 10 below. However, as
shown in Figures 5 and 6 no changes occurred in serum
' IL-1 and TNF and none of the patients experienced fever
or inflammatory reactions. The results are consistent
with the in vitro data reported in the earlier
examples.
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Table 8
Change In Absolute Neutrophil Counts (x 1000/1)
After Neutral Soluble Glucan Administration
Dose Level B Hour 8 Hour 12 Hour 24
Saline Mean 4.06 4.34 4.31 3.43
SD~ 2.12 1.53 1.16 1.46
N 6 6 6 6
IO 2.5 mg/kg Mean 4.11 11.29' 8.18 5.32
Neutral SDI 1.15 4.39 3.80 1.75
Soluble N' 6 6 6 6
Glucan
I5 B = Baseline a~easurE:ment
' p < 0.01 with respect to baseline
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Table 9
Change: in Monocyte Counts (X 1000/~C1)
After Soluble Administration
Neutral Glucan
Dose Level B Hour 8 Hour 12 Hour
24
Saline Mean 0.33 0.44 0.59 0.33
SD 0.09 0.10 0.22 0.12
N 6 6 6 6
2.5 mg/kg Mean 0.24 0.63' 0.67' 0.31
Neutral SD 0.10 0.24 0.32 0.15
Soluble N 6 6 6 6
Glucan
B = Baseline
measurement
p < 0.01 with respect baseline
to
Table 10
~x Vivo Microbicidal Activity of Nonaal Volunteers
Receiving Neutral Soluble Glucan
Mean Change in % Killing'
Dose Level Hour Hour 6 Hour 24 Day 2 Day 3 Day
3 6
Saline 0 0 0 0 0 0
2.5 mg/kg Mean 42.86 32.33 20.90 48.96 39.22 31.17
Neutral N 6 6 6 6 6 6
Soluble p-Value 0.062 0.036 0.300 0.045 0.085 0.026
Glucan
1 Normalized with respect to the saline control
-4 0- 214 2 811
EXAMPLE 9: ~MONSTRATION OF EFFICACY IN VIVO AS
OMAN ANTI- INFECTIVE '
In this clinical study, the safety, tolerance, and
potential e:Eficac.y of the neutral soluble ~-glucan was
evaluated in patients undergoing major thoracoabdominal
surgery with high risk of post-operative infection.
Thirty-four males and females who underwent surgery
received 0. l5 mg/k~g of the neutral soluble ~-glucan
preparation or saline placebo, given as an intravenous
infusion of 50 to 200 ml over one hour. Patients
received multiple sequential doses of the neutral
soluble ~-g:lucan or placebo at 12 to 24 hours prior to
surgery, 1 to 4 hours prior to surgery, 48 hours post-
surgery, an~3 96 hours post-surgery.
Hospitalization, infections, and usage of anti-
infective medications were examined as potential
clinical efficacy parameters. Compared to patients
given saline placebo infusions, patients who received
the neutral soluble ~=glucan spent an average of five
fewer days in the hospital (12.3 ~ 6.1 days versus 17.3
+ 15.5 days) and three fewer days in the Intensive Care
Unit (0.1 ~ 0.4 versus 3.3 + 6.3 days; p<0.03, one-way
analysis of variance).
The number o~f anti-infective medication prescrip-
tions written per study day following surgery was
consistently higher for control patients than for ~-
glucan recipient patients. Control patients were
prescribed an average of three times the number of
anti-infective medications as ~-glucan recipients over
the time period from surgery to discharge (p<0.005).
During the Treatment and Post-Treatment Follow-up
Phases, a total of 22 culture-confirmed infections in 5
control patients and 8 infections in 5 ~-glucan
recipient patienta were identified (p<0.002).
Neutrophils (PMNs) and monocytes/macrophages (MOs)
were purified from blood samples obtained at Baseline,
fB
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Day 1, and Day !i and examined for basal and phorbol
myrisate acetatc= stimulated microbicidal activity
against Etavhylc~coccus aureus, Escherichia coli and
Candida albicanaz. The neutral soluble ~-glucan
treatment gener~illy increased the basal and phorbol-
induced microbic:idal activity of MOs and PMNs.
EXAMPLE 10: W_OUtJD HEALING EFFECTS OF NEUTRAL
SOLZJBLE GLUCANS
Wound healing studies were performed in a hairless
mouse model hav:lng full thickness wounds with and
5 without Sta~h~lc~coccus aureus infection. Hairless SKH-
1 inbred mice (ci-8 weeks of age) were anesthetized with
ether and a mid:line 3 cm full thickness longitudinal
incision was made with a number 10 scalpel blade,
producing a full thickness wound that did not penetrate
the underlying ~Eascia. Incisions were closed using
steel clips placed at 1 cm intervals.
Formulations of neutral soluble glucan in phos-
phate buffered a~aline were applied 30 minutes following
wounding and re~spplied at 24 hour intervals during the
seven day post-operative period. Two micrograms of
neutral soluble glucan/mouse per day was topically
applied. Wound: were examined daily and rank-ordered
for effectiveness of formulation for enhancement of
visual based wound healing. Wounds were scored for
closure on a sc~ile of 0-5, with 5 indicating the most
healing. In on~~ graup of mice infected, the wound was
treated with a ~~ulture of 10' Staphylococcus aureus 30
minutes after w~~undxng and 2 hrs prior to treatment
with the neutra:L soluble glucan formulation.
Histologic~~l evaluation of the wound site of each
test group was ~nade» The dermis of the control group
(untreated wound) was heavily infiltrated with both
lymphocytes and monocytes/macrophages. However, re-
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epithelialization that occurred at the epidermal layer
was incomplete. The tissue section showed that the
dermal tissue was weak, in that the tissue integrity
was not maintained when it was sectioned.
The histology of the wounded tissue isolated from
mice treated for three days with phosphate buffered
saline containing the neutral soluble glucan showed
that there was a heavy infiltration of macrophages and
lymphocytes. Tissue integrity was good.
When topically applied to a wound, a composition
of neutral soluble glucan stimulated white blood cell
entry and activity at the wound site and accelerated
wound healing within the dermal layer of the wound.
Furthermore, the composition effectively eliminated
infection produced by bacterial infection (S,_. aureus)
and prevented the progression to sepsis. Untreated
wounds progressed to sepsis.
EXAMPLE 11: ,S'T'Tt~ttt~,TION OF PLATELET PROLIFERATION BY
~1EUTRAL SOLUBLE GLUCAN
The platelet proliferation stimulatory effect of
the neutral soluble glucan was tested in an animal
model system following either irradiation or
administration of the chemotherapeutic agent cisplatin.
These experiments demonstrated the unexpected platelet
stimulatory effect.
More specifically, saline or neutral soluble
glucan prepared as described in Example 1 was
administered to groups of 10 mice as a single IV bolus
20 hours prior to radiation exposure. Mice were
bilaterally exposed to a total-body irradiation of 7.5-
Gy. Fourteen days after irradiation the mice were
sacrificed and whole blood samples were analyzed for
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peripheral blood counts. As shown in Figure 7, the
platelet cell count from neutral soluble glucan-treated
mice was increased nearly 3-fold relative to saline-
treated control. levels.
In addition to tests on irradiated mice,
cisplatin-treated mice were also tested for the effect
of the neutral soluble glucan on platelet
hematopoiesis. Balb/c mice were injected intravenously
with cisplatin at a dose of 9.3 mg/kg through the tail
vein one hour lr~efore injecting either saline or the
neutral soluble glucan, prepared as described in
Example 1, intramuscularly in a single dose of 0
(saline) or 2 a~g/kg on Day 0. Platelet counts were
determined before treatment (Day 0) and at 2, 4, 6, 8,
and 10 days post-treatment. The results of this
experiment are shown in Figure 8. Each data point
represents the mean and standard error of platelet
counts from five mice. The statistically significant
differences (p<0.05) between the saline and neutral
soluble glucan (2 mg/kg) are noted.
Biological Depcs_it
Saccharomvces cerevisiae strain R4 Ad was
deposited on August 20, 1992 with the American Type
Culture Collection (ATCC), 12301 Parklawn Drive,
Rockville, Maryland, under the terms of the Budapest
Treaty. The strain has been assigned ATCC accession
number 74181. Upon issuance of a patent, this deposit
will be irrevocable.
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~cruivalents
Those skilled in the art will recognize or be able
to ascertain, using no more than routine
experimentation, many equivalents to the specific
materials and components described herein. Such
equivalents are intended to be encompassed in the scope
of the following claims:
