U.S. patent application number 10/719432 was filed with the patent office on 2004-06-17 for underivatized, aqueous soluble beta(1-3) glucan, composition and method of making same.
Invention is credited to Easson, D. Davidson JR., Jamas, Spiros, Ostroff, Gary R..
Application Number | 20040116380 10/719432 |
Document ID | / |
Family ID | 25464815 |
Filed Date | 2004-06-17 |
United States Patent
Application |
20040116380 |
Kind Code |
A1 |
Jamas, Spiros ; et
al. |
June 17, 2004 |
Underivatized, aqueous soluble beta(1-3) glucan, composition and
method of making same
Abstract
The present invention relates to neutral, aqueous soluble
.beta.-glucans which exert potent and specific immunological
effects without stimulating the production of certain cytokines, to
preparations containing the novel .beta.-glucans, and to a novel
manufacturing process therefor. The neutral, aqueous soluble
.beta.-glucan preparation has a high affinity for the .beta. glucan
receptor of human monocytes and retains two primary biological (or
immunological) activities, (1) the enhancement of microbicidal
activity of phagocytic cells, and (2) monocyte, neutrophil and
platelet hemo-poietic activity. Unlike soluble glucans described in
the prior art, the neutral, aqueous soluble .beta.-glucan of this
invention neither induces nor primes IL-1.beta. and TNF.alpha.
production in vitro and in vivo. Safe and efficacious preparations
of neutral, aqueous soluble .beta.-glucan of the present invention
can be used in therapeutic and/or prophylactic treatment regimens
of humans and animals to enhance their immune response, without
stimulating the production of certain biochemical mediators (e.g.,
IL-I.beta., TNF.alpha.) that can cause detrimental side effects,
such as fever and inflammation.
Inventors: |
Jamas, Spiros; (Boston,
MA) ; Easson, D. Davidson JR.; (Shrewsbury, MA)
; Ostroff, Gary R.; (Worcester, MA) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Family ID: |
25464815 |
Appl. No.: |
10/719432 |
Filed: |
November 21, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10719432 |
Nov 21, 2003 |
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09841179 |
Apr 24, 2001 |
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09841179 |
Apr 24, 2001 |
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09326513 |
Jun 4, 1999 |
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09326513 |
Jun 4, 1999 |
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09166454 |
Oct 5, 1998 |
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09166454 |
Oct 5, 1998 |
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08469233 |
Jun 6, 1995 |
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5817643 |
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09166454 |
Oct 5, 1998 |
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08373251 |
Jan 26, 1995 |
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5783569 |
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08373251 |
Jan 26, 1995 |
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PCT/US93/07904 |
Aug 20, 1993 |
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08373251 |
Jan 26, 1995 |
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07934015 |
Aug 21, 1992 |
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5622939 |
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Current U.S.
Class: |
514/54 |
Current CPC
Class: |
A61P 37/04 20180101;
A61P 17/02 20180101; Y02A 50/30 20180101; A61P 31/04 20180101; A61P
31/12 20180101; A61P 43/00 20180101; A61P 37/00 20180101; A61K
31/715 20130101; A61P 7/00 20180101; A61P 31/00 20180101; C08B
37/0024 20130101 |
Class at
Publication: |
514/054 |
International
Class: |
A61K 031/715 |
Claims
What is claimed is:
1. A neutral, aqueous soluble .beta.-glucan preparation which
enhances host defense mechanisms to infection and does not induce
an inflammatory response.
2. An underivatized, aqueous soluble .beta.-glucan composition in a
triple helix conformation which enhances host immune defense
mechanisms without inducing detrimental side effects.
3. An underivatized, aqueous soluble .beta.-glucan composition in a
triple helix conformation having a high affinity to the glucan
receptor on monocytes and which enhances microbicidal activity of
phagocytic cells and the hemopoietic activity of monocytes,
neutrophils and platelets.
4. An underivatized, aqueous soluble .beta.-glucan composition in a
triple helix conformation which is effective in mobilizing the
hosts normal immune defenses.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
09/841,179, filed Apr. 24, 2001, which is a continuation of
application Ser. No. 09/326,513, filed Jun. 4, 1999, which is a
continuation of application Ser. No. 09/166,454, filed Oct. 5,
1998, which is a continuation of application Ser. No. 08/469,233,
filed Jun. 6, 1995 (now U.S. Pat. No. 5,817,643), and application
Ser. No. 08/373,251 filed Aug. 20, 1993 (now U.S. Pat. No.
5,783,569), which is the U.S. National stage of International
Application No. PCT/US93/07904, filed Aug. 20, 1993, which is a
continuation-in-part of application Ser. No. 07/934,015 filed Aug.
21, 1992 (now U.S. Pat. No. 5,622,939). The entire teachings of the
above applications are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] In the early 1960's, zymosan, 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, to promote survival
following irradiation, to increase resistance to bacterial
infections, to inhibit tumor development, to promote graft
rejection, and to inhibit dietary-induced hypercholestarolemia 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 .beta.-glucan. In conventional
nomenclature, the polysaccharide .beta.-glucan is known as
poly-(1-6)-.beta.-O-glucopyranosyl-(1-3)-.beta.-D-glucopyranose
(PGG). Repetition of biological assays with .beta.-glucan indicated
that most of the above functional activities identified with
zymosan were retained by the purified .beta.-glucan
preparation.
[0003] The properties of .beta.-glucan are quite similar to those
of endotoxin in increasing nonspecific immunity and resistance to
infection. The activities of .beta.-glucan as an immune adjuvant
and hemopoietic stimulator compare to those of more complex
biological response modifiers (BRMs), such as bacillus
Calmette-Guerin (BCG) and Corynebacterium parvum. The functional
activities of yeast .beta.-glucan are also comparable to those
structurally similar carbohydrate polymers isolated from fungi and
plants. These higher molecular weight (1-3) -.beta.-D-glucans such
as schizophyllan, lentinan, krestin, grifolan, and pachyman exhibit
similar immunomodulatory activities. A common mechanism shared by
all these .beta.-glucan preparations is their stimulation of
cytokines such as interleukin-1 (IL-1) and tumor necrosis factor
(TNF). Lentinan has been extensively investigated for its antitumor
properties, both in animal models 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 per week alone, or in
combination with antineoplastic drugs. In addition to the
activities ascribed to yeast glucans, studies suggest lentinan acts
as a T-cell immunopotentiator, inducing cytotoxic activities,
including production of interleukins 1 and 3 and colony-stimulating
factors (CSF). (Chihara et al., 1989, Int. J. Immunotherapy,
4:145-154; Hamuro and Chihara, In Lentinan, An
Immunopotentiator)
[0004] Various preparations of both particulate and soluble
.beta.-glucans have been tested in animal models to evaluate
biological activities. The use of soluble and insoluble
.beta.-glucans alone or as vaccine adjuvants for viral and
bacterial antigens has been shown in animal models to markedly
increase resistance to a variety of bacterial, fungal, protozoan
and viral infections. The hemopoietic effects of .beta.-glucan have
been correlated with increased peripheral blood leukocyte counts
and bone marrow and spleni ccellularity, reflecting increased
numbers of granulocyte-macrophage 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 hemopoietic and anti-infective
effects of .beta.-glucan were active in cyclophosphamide-treated
immunosuppressed animals. .beta.-glucan was shown to be beneficial
in animal models of trauma, wound healing and tumorigenesis.
However, various insoluble and soluble preparations of
.beta.-glucan differed 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 (DiLuzio et al., 1979, Int.
J. of Cancer, 24:773-779; DiLuzio, U.S. Pat. No. 4,739,046).
[0005] The molecular mechanism of action of .beta.-glucan has been
elucidated by the demonstration of specific .beta.-glucan receptor
binding sites on the cell membranes of human neutrophils and
macrophages. Mannans, galactans, .alpha.(1-4)-linked glucose
polymers and .beta.(1-4)-linked glucose polymers have no avidity
for this receptor. These .beta.-glucan binding sites are
opsonin-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 .beta.-glucan receptor, in the absence of antibody,
results in complement activation, phagocytosis, lysosomal enzyme
release, and prostaglandin, thromboxane and leukotriene generation;
thereby increasing nonspecific resistance to infection. However,
soluble .beta.-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 in vivo and corresponded to induction of the
synthesis of these cytokines in vitro. (See Sherwood et al., 1987,
Int. J. Immunopharmac., 9:261-267 (enhancement of IL-1 and IL-2
levels in rats injected with soluble glucan); Williams et al.,
1988, Int. J. Immunopharmac., 10:405-414 (systemic administration
of soluble glucan to AIDS patients increased IL-1 and IL-2 levels
which were accompanied by chills and fever); Browder et al., 1990,
Ann. Surg., 211:605-613 (glucan administration to trauma patients
increased serum IL-1 levels, but not TNF levels); Adachi et al.,
1990, Chem. Pharm. Bull., 38:988-992 (chemically cross-linked
.beta.(1-3) glucans induced IL-1 production in mice).)
[0006] 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 et al., J.
Parent. Ent. Nutr. 12(2):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 al., Ann.
Int. Med., 106:421-433 (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 excretion,
anorexia, slow wave sleep, bone resorption, decreased pain
threshold and expression of many inflammatory-associated cytokines.
It is also toxic to the insulin secreting beta cells of the
pancreas. Patients suffering from a number of inflammatory diseases
have elevated levels of IL-1 in their systems. Administration of
agents that enhance further IL-1 production only exacerbate these
inflammatory conditions.
[0007] Tumor necrosis factor is also involved in infection,
inflammation and cancer. Small amounts of TNF release growth
factors while 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
[0008] The present invention relates to neutral soluble
.beta.-glucans which enhance a host's immune defense mechanisms to
infection but do not induce an inflammatory response, to
preparations containing the neutral soluble .beta.-glucans, and to
a novel manufacturing process therefor. In the present method,
soluble glucan which induces cytokine production is processed
through a unique series of acid, alkaline and neutral treatment
steps to yield a conformationally pure neutral soluble glucan
preparation with unique biological properties. The neutral soluble
glucan preparation retains a specific subset of immunological
properties common to .beta.-glucans but uniquely does not induce
the production of IL-1 and TNF in vitro or in vivo. Throughout this
specification, unless otherwise indicated, the expressions "neutral
soluble glucan" and "neutral soluble .beta.-glucan" refer to the
composition prepared as described in Example 1.
[0009] The neutral soluble glucan preparation is produced by
treating insoluble glucan 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, and further purifying under neutral pH
to remove single helix and aggregated materials to yield a
conformationally pure, neutral, water soluble, underivatized glucan
which has a unique biological profile.
[0010] The neutral soluble glucan preparation has a high affinity
for the .beta.-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 in vitro and in vivo.
[0011] 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 immune 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 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
[0012] FIG. 1 shows the general structure of neutral soluble glucan
as being a linear .beta.(1-3)-linked glucose polymer having
periodic branching via a single .beta.(1-6)-linked glucose
moiety.
[0013] FIG. 2 shows a gel permeation chromatogram (pH 7) of soluble
glucan which has not been purified by alkali dissociation and
re-annealing. The chromatogram shows three species, referred to
herein as high molecular weight aggregate (Ag), Peak A and Peak B
(single helix glucan).
[0014] FIG. 3 is a chromatogram obtained for the neutral soluble
glucan by gel permeation chromatography. The solid line represents
the neutral soluble glucan at pH 7 and the broken line represents
the neutral soluble glucan at pH 13.
[0015] FIG. 4 is a chromatogram obtained for the single helix
.beta.-glucan (Peak B) by gel permeation chromatography. The solid
line represents Peak B at pH 7 and the broken line represents Peak
B at pH 13.
[0016] FIG. 5 shows the change in serum IL-1 levels, over time,
taken from patients intravenously infused with placebo (broken
line) or neutral soluble glucan (solid line).
[0017] FIG. 6 shows the change in serum TNF levels, over time,
taken from patients intravenously infused with placebo (broken
line) or neutral soluble glucan (solid line).
[0018] FIG. 7 is a diagram representing peripheral blood counts
from irradiated mice following administration of neutral soluble
glucan.
[0019] FIG. 8 is a diagram representing platelet cell counts from
cisplatin-treated mice following administration of neutral soluble
glucan.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The invention relates to a neutral soluble .beta.-glucan
polymer that can bind to the .beta.-glucan receptor and activate
only a desired subset of immune responses. The terms "neutral
soluble .beta.-glucan" and "neutral soluble glucan", unless
otherwise specified, refer to the composition prepared as described
in Example 1.
[0021] This neutral soluble .beta.-glucan has been shown to
increase the number of neutrophils and monocytes as well as their
direct infection fighting activity (phagocytosis and microbial
killing). However, the neutral soluble .beta.-glucan does not
stimulate the production of biochemical mediators, such as IL-1 and
TNF, that can cause detrimental side effects such as high fever,
inflammation, wasting disease and organ failure. These advantageous
properties make neutral soluble glucan preparations of this
invention useful in the prevention and treatment of infection
because they selectively activate only those components of the
immune system responsible for the initial response to infection,
without stimulating the release of certain biochemical mediators
that can cause adverse side effects. The solution containing the
neutral soluble .beta.-glucan also lacks the toxicity common to
many immunomodulators.
[0022] The neutral soluble .beta.-glucans of this invention are
composed of glucose monomers organized as a .beta.(1-3) linked
glucopyranose backbone with periodic branching via .beta.(1-6)
glycosidic linkages. The neutral soluble glucan preparations
contain glucans, which have not been substantially modified by
substitution 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 .beta.-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.
[0023] The neutral soluble glucan preparations of this invention
are prepared from insoluble glucan particles, preferably derived
from yeast organisms. See Manners et al., Biochem. J., 135:19-30,
(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 al., in U.S. Pat. Nos. 4,810,646, 4,992,540,
5,082,936 and 5,028,703, the teachings of all 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 which contain .beta.-glucans in their cell walls. Whole
glucan particles obtained from the strains Saccharamyces cerevisiae
R4 (NRRL Y-15903; deposit made in connection with U.S. Pat. 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 microellipsodes,
Saccharomyces carlsbergensis, Schizosacharomyces pombe,
Kluyveromyces lactis, Kluyveremvcesfragilis, Kluyveromyces
polysporus, Candida albicans, Candida cloacae, Candida tropicalis,
Candida utilis, Hansenula wingeri, Hansenula arni, Hansenula
henricii, Hansenula americana.
[0024] A procedure for extraction of whole glucan particles is
described by Jamas et al., in U.S. Pat. Nos. 4,810,646, 4,992,540,
5,082,936 and 5,028,703. For the purpose of this present invention,
it is not necessary to conduct the final organic extraction and
wash steps described by Jamas et al.
[0025] In the present 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 100.degree. C. is sufficient. Preferably, the
acid used is an organic acid capable of dissolving the acid-soluble
glucan 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/v) are useful for this purpose. The treatment time may vary from
about 10 minutes to about 20 hours depending on the acid
concentration, temperature and source of whole glucan particles.
For example, modified glucans having more .beta.(1-6) branching
than naturally occurring, or wild-type glucans, require more
stringent conditions, i e., longer exposure times and higher
temperatures. This acid-treatment step can be repeated under
similar or variable conditions. One preferred processing method is
described in the exemplification using glucan derived from S.
cerevisiae strain R4 Ad. In another embodiment of the present
method, whole glucan particles from the strain, S. cerevisiae R4,
which have a higher level of .beta.(1-6) branching than
naturally-occurring glucans, are used, and treatment is carried out
with 90% (w/v) formic acid at 20.degree. C. for about 20 minutes
and then at 85.degree. C. for about 30 minutes.
[0026] 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 slurry is
adjusted with an alkaline compound such as sodium hydroxide, to a
pH of about 7 to about 14. The precipitate is collected by
centrifugation and is boiled in purified water (e.g., USP) for
three hours. The slurry is then resuspended in hot alkali having a
concentration sufficient to solubilize the glucan polymers.
Alkaline compounds which can be used in this step include
alkali-metal 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.degree. C. to about 121.degree. C.,
preferably from about 20.degree. C. to about 100.degree. 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.degree. C. and a contact time of approximately 1-2 hours.
The resulting mixture contains solubilized glucan molecules and
particulate glucan residue and generally has a dark brown color due
to oxidation of contaminating 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 volumes of ethanol. The mixture is chilled to about
4.degree. 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.degree. C.
and 100.degree. C. At this point the pH is adjusted to
approximately 10 to 13 with a base such as sodium hydroxide.
[0027] 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.
[0028] 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.degree.
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
ordered triple helical conformation. The re-annealed glucan
solution is then size fractionated, for example by using
30,000-70,000 NMW and 100,000-500,000 NMW cut-off membrane
ultrafilters 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
aggrecgates, 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 ultrafilters are used is a matter of preference.
[0029] The concentrated fraction obtained is enriched in the
soluble, biologically 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.
[0030] The neutralized solution can then be further purified, for
example, by diafiltration, using a pharmaceutically acceptable
medium (e.g., sterile water for injection, phosphate-buffered
saline (PBS), isotonic saline, dextrose) suitable for parenteral
administration. The preferred membrane 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 .mu.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.degree. C.) for extended periods
of time without degradation. This process is unique in that it
results in a neutral aqueous solution of (pH 4.5 to 7.0)
immunologically active glucans which is suitable for parenteral
administration.
[0031] 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.degree. 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 "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.
[0032] 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.
[0033] 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 (e.g., IL-1, TNF,
IL-6 IL-8 and GM-CSF) that can cause adverse side effects. As such,
the present soluble glucan composition can be used to prevent or
treat infectious diseases in malnourished 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 of who may have weakened immune
systems. An immunocompromised 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.
[0034] 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 immune response to be reduced or depressed, such
as HIV infection (AIDS). For example, the method can be used to
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 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.
[0035] 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, parenteral, 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.
[0036] In general, the compositions of the present invention can be
administered to an individual periodically as necessary to
stimulate the individual's immune response. An individual skilled
in the medical arts will be able to determine the length 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.
[0037] Neutral soluble .beta.-glucan can be used for the prevention
and treatment of infections caused by a broad spectrum of
bacterial, fungal, viral and protozoan pathogens. The prophylactic
administration of neutral soluble .beta.-glucan 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,
transurethral prostatectomy) and in patients in whom infection at
the operative site would present a serious risk (e.g., prosthetic
arthroplasty, cardiovascular surgery), concurrent initial therapy
with an appropriate antibacterial agent and the present neutral
soluble glucan preparation will reduce the incidence and severity
of infectious complications.
[0038] In patients who are immunosuppressed, not only by disease
(e.g., cancer, AIDS) but by courses of chemotherapy and/or
radiotherapy, the prophylactic administration of the soluble glucan
will reduce the incidence of infections caused by a broad spectrum
of opportunistic pathogens including many unusual bacteria, fungi
and viruses. Therapy using neutral soluble .beta.-glucan has
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.
[0039] 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 .beta.-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 antimicrobial agents
and the neutral soluble .beta.-glucan is indicated for the
treatment of chronic, severe, refractory, complex and difficult to
treat infections.
[0040] The compositions administered in the method of the present
invention can optionally include other components, in addition to
the neutral soluble .beta.-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 .beta.-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
.beta.-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, 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.
[0041] 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 process. Alternatively, the neutral soluble
.beta.-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.
[0042] Another particular use of the compositions of this invention
is for the treatment of myelodysplastic syndrome (MDS). MDS,
frequently referred to as preleukemia syndrome, is a group 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 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.
[0043] 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 .beta.-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.
[0044] 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 disclosed platelet stimulating property had
not been reported or anticipated. 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 (reduced polymorphonuclear (PMN)
leukocyte cell count) and thrombocytopenia (reduced platelet
count). At present, these conditions are treated by the
administration of colony-stimulating factors such as GM-CSF and
granulocyte colony-stimulating factor (G-CSF). Such factors are
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 agent to prevent or minimize the
development of thrombocytopenia which limits the dose of the
radiation or chemotherapeutic agent which is used to treat
cancer.
[0045] The invention is further illustrated by the following
Examples.
EXAMPLES
Example 1
Preparation of Neutral Soluble Glucan from S. Cerevisiae
[0046] Saccharomvces cerevisiae strain R4 Ad (a non-recombinant
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-batchmode (New Brunswick MPP80). When the
growing culture reached late logarithmic phase, the fermentation
was ended and the .beta.-glucan was stabilized by adjusting the
culture to pH 12.+-.0.5 using 10 M NaOH. The yeast cells containing
.beta.-glucan were harvested by continuous-flow centrifugation
(Westfalia SA-1). After centrifugation, the cells were collected
into a stainless steel extraction vessel.
[0047] 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.degree. C. for 1 hour. Upon completion
of this alkaline extraction, the .beta.-glucan remained in the
solid 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 deacetylated chitin to chitosan
in the cell wall.
[0048] The second step in the extraction process was a pH
4.5.+-.0.05 (adjusted with concentrated HCl) extraction at
75.+-.5.degree. C. for 1 hour. This was followed by a 0.1 M acetic
acid extraction to complete the removal of glycogen, chitin,
chitosan and remaining proteins. The solids were collected and
rinsed twice with Purified Water USP to remove any residual acid as
well as any yeast degradation products.
[0049] The third step in the extraction process was a set of six
organic extractions. The first four extractions were carried out in
isopropanol. The solids were collected by centrifugation and then
subjected to two acetone extractions. The two-stage organic
extractions eliminated nonpolar lipids and hydrophobic proteins
which may have co-purified with the drug substance. The resulting
wet solids were dried in a vacuum oven at 65.+-.5.degree. C. for
48-96 hours to yield a free-flowing powder.
[0050] At this stage the extraction process yielded a stable,
insoluble intermediate consisting of approximately 90%
.beta.-glucan, called whole glucan particles (WGPs). The dry WGP
intermediate was stored at 15-30.degree. C. until further use.
[0051] The WGP powder was resuspended in 98% (w/v) formic acid, in
a glass reaction vessel at room temperature. The resulting mixture
was heated to 85.+-.5.degree. C. for 20 minutes. Under these
conditions, the WGPs were partially hydrolyzed and solubilized to
provide the desired molecular weight distribution of soluble
.beta.-glucan which was then precipitated by adding 1.5 volumes of
ethanol. After complete mixing, the preparation was centrifuged to
collect the .beta.-glucan precipitate. Any residual formic acid was
removed by boiling the .beta.-glucan preparation in Purified Water
USP for three hours.
[0052] Any unhydrolyzed WGPs were then removed from the
.beta.-glucan solution by centrifugation. The .beta.-glucan
solution was raised to pH 12.5.+-.0.5 by the addition of the
concentrated sodium hydroxide. The remaining purification steps
were carried out by ultrafiltration.
[0053] The soluble alkaline .beta.-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
.beta.-glucan. Trace lows 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.
[0054] The .beta.-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.degree. 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.
[0055] The neutral soluble glucan was then concentrated in a 10,000
NMW cut-offf membrane ultrafilter. The concentration process
continued until a concentration of at least 1.0 mg/ml hexose
equivalent was achieved.
[0056] 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 glucan. The neutral soluble glucan
solution was stored at controlled room temperature (15-30.degree.
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
solubility increased with increasing pH and reached approx. 150
mg/ml at pH 13.
Example 2
Analysis of Neutral Soluble Glucan
[0057] A. Glucose, Mannose and Glucosamine
[0058] Monosaccharide analysis was performed to quantitate the
relative amounts of .beta.-glucan (as glucose), mannan or
phosphomannan (as mannose), and chitin (as N-acetyl glucosamine) in
the neutral soluble glucan. The sample was hydrolyzed to
monosaccharides in 2 M trifluoroacetic acid for 4 hours at
110.degree. C., evaporated to dryness, and redissolved in water.
Monosaccharides were separated on a Dionex HPLC system using a
CarboPac PA100 column (4.times.250 mm) using 5 M NaOH at 1 ml/min
and quantitated using a pulsed electrochemical detector (Dionex
Model PED-1). The sensitivity of this assay for monosaccharides is
0.1% (w/w).
[0059] Glucose (retention time of 16.6 min) was identified as the
only monosaccharide component of neutral soluble glucan along with
traces of glucose degradation products (from hydrolysis)
anhydroglucose at 2.5 min and 5-hydroxymethylfurfural at 4.3 min.
The results confirm that neutral soluble glucan consisted of
.gtoreq.98% glucose.
[0060] B. FTIR
[0061] Fourier transform infrared spectroscopy by diffuse
reflectance (FTIR, Matson Instruments, Polaris) of lyophilized
neutral soluble glucan samples was used to determine the anomeric
structure (.alpha. vs. .beta.), and linkage type (.beta.(1-3),
.beta.(1-6), .beta.(1-4)) present in neutral soluble glucan.
Absorption maxima of 890 cm.sup.-1 identified .beta.(1-3) linkages;
920 cm.sup.-1 identified .beta.(1-6) linkages. No presence of
.alpha.-linked anomers (e.g., glycogen, 850 cm.sup.-1) or
.beta.(1-4)-linked polysaccharides (e.g., chitin, 930 cm.sup.-1)
were detected.
Example 3
Conformational Analysis
[0062] A solution of .beta.-glucan which was not processed by
alkali dissociation and re-annealing was analyzed for its
compositional identity by gel permeation chromatography (pH 7) and
found to contain multiple species, referred to herein as high
molecular weight aggregate (Ag), Peak A and Peak B (See FIG. 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 FIG. 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 underwent a significant conformational
transition from pH 7 to pH 13 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.
1 TABLE 1 MW MW Ratio Sample 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
[0063] The conformation of neutral soluble glucan and Peak B glucan
was also determined by aniline blue complexing (Evans et al., 1984,
Carb. Pol., 4:215-230; Adachi et al., 1988, Carb. Res.,
177:91-100), using curdlan, a linear .beta.(1-3) glucan as the
triple helix control and pustulan, a .beta.(1-6) glucan, as a
non-ordered conformational control. The results are discussed below
and shown in Table 2.
[0064] 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 resulting in low fluorescence
measurements which were not affected by NaOH concentration.
[0065] The neutral soluble glucan complexed effectively with
aniline blue at low NaOH concentration (25 mM NaOH) resulting in
high fluorescence. However, the neutral soluble glucan conformation
dissociated significantly (50%) at NaOH concentrations as low as
150 mM NaOH indicating that it exists as a unique conformation
compared to naturally occurring .beta.-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.
2TABLE 2 Conformational Analysis of Glucans by Aniline Blue
Complexing Fluorescence 25 mM 100 mM 150 mM Test Material NaOH NaOH
NaOH Blank 0 2 0 Curdlan 53.5 41.6 36 .beta.(1-3) glucan Pustulan
9.8 8.3 8.0 .beta.(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
TNF.alpha.
[0066] Human peripheral blood mononuclear cells were isolated
(Janusz et al., (1987), J. Immunol., 138: 3897-3901) 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, N.J.). The mononuclear cells
were collected into HBSS, washed twice, resuspended in RPMI 1640
Medium (Gibco, Grand Island, N.Y.) containing 1% heat-inactivated
autologous serum (55.degree. C. for 30 min.), and counted on the
Coulter counter.
[0067] For the preparation of monocyte monolayers, 1 ml of
2.2.times..10.sup.6 mononuclear cells/ml was plated into wells of
24-well tissue culture plates (CoStar, Cambridge, Mass.), incubated
for 1 hour at 37.degree. C. in a humidified atmosphere of 5%
CO.sub.2, and washed three times with RPMI to remove nonadherent
cells. A second 1 ml aliquot of 2.2.times.10.sup.6 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 40.times. with an inverted phase microscope and a
calibrated reticle, the number of adherent cells for 30 different
donors was 0.77.+-.0.20.times.10.sup.6 per well (mean .+-.SD). By
morphology and nonspecific esterase staining, >95% of the
adherent cells were monocytes.
[0068] Monocyte monolayers were incubated at 37.degree. C. in the
CO.sub.2 chamber for 0 to 8 hours with 0.5 ml of RPMI, 1% heat
inactivated autologous serum, 10 mM HEPES, and 5 mM MgCl.sub.2 in
the absence and presence of various glucan preparations. The
culture supernatant was removed, clarified by centrifugation at
14,000 g for 5 min at 4.degree. C., and stored at -70.degree. C.
before assay of TNF.alpha..
[0069] The concentration of TNF.alpha. in the monocyte supernatants
was measured by an enzyme-linked immunoadsorbent assay (ELISA) with
the BIOKINE TNF Test kit (T Cell Sciences, Cambridge, Mass.), which
had a lower limit of detectability of 40 pg/ml. The data are
expressed as pg per 10.sup.6 monocytes, `which was calculated by
dividing the quantity of cytokine in 0.5 ml of supernatant by the
number of monocytes per well.
[0070] For the determination of cell-associated levels of
TNF.alpha., 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.degree. C. and
the resulting supernatants were stored at -70.degree. C. Newly
prepared monocyte monolayers contained no detectable levels of
intracellular TNF.alpha..
[0071] The results are shown in Tables 3 and 4 below.
3TABLE 3 TNF.alpha. Synthesis by Human Monocytes Stimulated with
Various Glucan Preparations TNF.alpha. (pg/10.sup.6 monocytes)
Glucan Conc. 1 2 3 Mean .+-. SD Buffer Control 36 39 2 26 .+-. 21
Neutral soluble 1 mg/ml 44 51 33 43 .+-. 9 glucan Laminarin 1 mg/ml
372 324 227 308 .+-. 74 Whole 4 .times. 10.sup.7/ml 2129 1478 1683
1763 .+-. 333 Glucan particles
[0072]
4TABLE 4 TNF.alpha. Stimulation by Different Conformational
Structures of Soluble .beta.-Glucan TFN.alpha. Glucan Conc.
(pg/10.sup.6 moncytes) 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
Particles 4 .times. 10.sup.7/ml 2065
[0073] Table 3 shows that TNF.alpha. was stimulated by insoluble
glucan particles and by laminarin, a soluble .beta.(1-6) and
.beta.(1-3) linked glucan. There was no stimulation of TNF.alpha.
by neutral soluble glucan. Table 4 shows similar results, but
further confirms that TNF.alpha. stimulation is dependent upon
conformational structure. The neutral soluble glucan did not
stimulate TNF.alpha. while Peak B (single helical conformation) did
stimulate TNF.alpha..
Example 5
Avidity of Neutral Soluble Glucan for the Glucan Receptor
[0074] Monolayers of human monocytes, prepared on siliconized glass
coverslips (Czop et al., 1978, J. Immunol., 120:1132), were
incubated for 18 minutes at 37.degree. C. in a humidified 5%
CO.sub.2 incubator with either 0.25 ml of buffer (RPMI-Mg-HEPES) or
a range of concentrations (0.1-50 .mu.g/ml) of neutral soluble
glucan. The monocyte monolayers were then washed twice with 50 ml
of RPM 1640 medium and were layered with 0.25 ml of
4.8.times.10.sup.6/ml zymosan particles (Czop and Austen, 1985, J.
Immunol., 134:2588-2593). After a 30 minute incubation at
37.degree. 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 1000.times.
light microscope.
[0075] Monocyte monolayers pretreated with buffer, 50 or 500
.mu.g/mi of neutral soluble glucan as described above were
subsequently tested for their capacity to ingest IgG coated sheep
erythrocytes (E.sup.SIgG). After an 18 minute preincubation with
the neutral soluble glucan, the monolayers were incubated with 0.25
ml of 1.times.107/ml E.sup.SIgG for 30 minutes at 37.degree. C.,
washed three times with 50 ml of Hank's balanced salt solution,
treated for 4 minutes with 0.84% NH.sub.4Cl to lyse noningested
E.sup.SIgG, and fixed and stained as described above. The
percentages of monocytes ingesting .gtoreq.1 and .gtoreq.3
E.sup.SIgG were determined by counting at least 300 monocytes per
monolayer.
[0076] The percent inhibition of monocyte ingestion was determined
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.
5TABLE 5 Glucan-receptor Binding Capacity of Distinct Conformations
of Soluble .beta.-glucans Test Materials Conc. % Inhibition Buffer
-- 0% Neutral soluble glucan 50 .mu.g/ml 74% 500 .mu.g/ml 86% Peak
B 50 .mu.g/ml 50% 500 .mu.g/ml 56%
[0077] Both .beta.-glucan preparations tested above inhibited
monocyte ingestion of zymosan particles demonstrating their
capacity to competitively bind to the .beta.-glucan receptor on
human monocytes. Neutral soluble glucan demonstrated a higher
receptor binding capacity than Peak B as indicated by the greater
level of inhibition achieved at both 50 .mu.g/ml and 500 .mu.g/ml.
This biological assay demonstrates that the neutral soluble glucan
is a superior ligand for the .beta.-glucan receptor.
Example 6
Lack of In Vitro Stimulation of IL-1.beta. and TNF.alpha. from
Human Mononuclear Cells
[0078] Venous blood was obtained from healthy male volunteers and
mononuclear cells were fractionated by Ficoll-Hypaque
centrifugation. The mononuclear cells were washed, resuspended in
endotoxin-free RPMI-1640 culture medium--ultrafiltered to remove
endotoxins as described elsewhere (Dinarello et al., 1987, J. Clin.
Microbiol. 25:1233-8)--at a concentration of 5.times.10.sup.6
cells/ml and were aliquoted into 96-well microtiter plates (Endres
et al., 1989, N.E. J. Med., 320:265-271). The cells were then
incubated with either 1 ng/ml endotoxin (lipopolysaccharide, E.
coli 055:B5, Sigma, St. Louis), or 10 to 1000 ng/ml .beta.-glucan,
at 37.degree. C. for 24 hours in 5% CO.sub.2 and then lysed by
three freeze-thaw cycles (Endres et al., 1989, N.E. J. Med.
320:265-271). Synthesis of IL-1.beta. and TNF.alpha. was determined
by specific radioimmunoassays as described elsewhere (Lisi et al.,
1987, Lymph Res. 6:229-244; Lonnemann et al., 1988, Lymph. Res.
7:75-84; Van der Meer et al., 1988, J. Leukocycte Biol.
43:216-223.
[0079] To determine if neutral soluble glucan could acts 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.degree.
C. in 5% CO.sub.2. The cells were washed to remove neutral soluble
glucan and were then incubated with 1 ng/ml endotoxin as described
above. IL-1.beta. and TNF.alpha. were determined as described
above.
[0080] 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.beta. or TNF.alpha. synthesis over
the control buffer treated cells. Endotoxin LPS, a known stimulant,
resulted in significantly 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 neutral soluble glucan (10-1000
ng/ml) and were then exposed to endotoxin as a co-stimulant.
Neutral soluble glucan did not result in any amplification of the
IL-1.beta. and TNF.alpha. levels compared to endotoxin alone.
6TABLE 6 In Vitro IL-1.beta. and TNF.alpha. Synthesis by Human
Peripheral Blood Mononuclear Cells IL-1.beta. TNF.alpha. Stimulant
(ng/ml).sup.1 (ng/ml).sup.1 Cells only <0.10 0.14 Neutral 10
ng/ml 0.13 0.16 soluble glucan 100 ng/ml 0.12 0.16 1000 ng/ml
<0.10 0.14 LPS 1 ng/ml 2.62 2.22 LPS (1 ng/ml) + Neutral 10
ng/ml 2.62 2.25 soluble 100 ng/ml 2.57 2.07 glucan 1000 ng/ml 2.85
2.27 .sup.1Values are the mean of two donors.
Example 7
In Vivo Protection Against Infection in Mice
[0081] A sepsis model was developed in rats to characterize the
efficacy of .beta.-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., 10: 1256-1259).
[0082] Groups of rats received neutral soluble glucan (100
.mu.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 blood and peritoneal cavity culminating in mortality. The cecal
inoculum contained an array of facultative species, such E. coli,
as well as other obligate anaerobes (Streptococcus sp., Bacteroides
sp., Clostridium perfringens, Clostridium ramosum,
Pentostreptococus magnus and productus, Proteus, mira-bilis). The
animals were observed four times per day for the first 48 h and
twice per day thereafter. The results are reported in Table 7.
7TABLE 7 Effect of Neutral Soluble Glucan on Mortality in a Rat
Model for Intra-abdominal Sepsis Group Mortality(%) P vs. Saline
Saline 12/20 (60) Neutral soluble glucan 2/10 (10) <0.01
[0083] These results demonstrate that neutral soluble glucan which
does not induce IL-1.beta. and TNF.alpha. protects mice from lethal
bacterial challenge.
Example 8
Demonstration of Safety for Human Administration
[0084] 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, IL6,
IL-8 and GM CSF. Single intravenous administration of neutral
soluble glucan resulted in an increase in monocytes and neutrophils
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 FIGS. 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.
8TABLE 8 Change in Absolute Neutrophil Counts (.times.1000/.mu.l)
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 2.5 mg/kg Mean 4.11 11.29* 8.18 5.32 Neutral SD 1.15
4.39 3.80 1.75 Soluble N 6 6 6 6 Glucan B = Baseline measurement *p
< 0.01 with respect to baseline
[0085]
9TABLE 9 Change in Monocyte Counts (.times.1000/.mu.l) After
Soluble Neutral Glucan Administration 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 to baseline
[0086]
10TABLE 10 Ex Vivo Microbicidal Activity of Normal Volunteers
Receiving Neutral Soluble Glucan Mean Change in % Killing.sup.1
Dose Level Hour 3 Hour 6 Hour 24 Day 2 Day 3 Day 6 Saline 0 0 0 0 0
0 2.5 Mean 42.86 32.33 20.90 48.96 39.22 31.17 mg/kg Neutral N 6 6
6 6 6 6 Soluble p- 0.062 0.036 0.300 0.045 0.085 0.026 Glucan Value
.sup.1Normalized with respect to the saline control
Example 9
Demonstration of Efficacy In Vivo as Human Anti-Invective
[0087] In this clinical study, the safety, tolerance, and potential
efficacy of the neutral soluble .beta.-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 0.5 mg/kg of the neutral soluble
.beta.-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
.beta.-glucan or placebo at 12 to 24 hours prior to surgery, 1 to 4
hours prior to surgery, 48 hours post-surgery, and 96 hours
post-surgery.
[0088] 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 .beta.-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).
[0089] The number of anti-infective medication prescriptions
written per study day following surgery was consistently higher for
control patients than for .beta.-glucan recipient patients. Control
patients were prescribed an average of three times the number of
anti-infective medications as .beta.-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 .beta.-glucan recipient patients were identified
(p<0.002).
[0090] Neutrophils (PMNs) and monocytes/macrophages (MOs) were
purified from blood samples obtained at Baseline, Day 1, and Day 5
and examined for basal and phorbol myrisate acetate stimulated
microbicidal activity against Staphylococcus aureus, Escherichia
coli and Candida albicans. The neutral soluble .beta.-glucan
treatment generally increased the basal and phorbolinduced
microbicidal activity of MOs and PMNs.
Example 10
Wound Healing Effects of Neutral Soluble Soluble Glucans
[0091] Wound healing studies were performed in a hairless mouse
model having full thickness wounds with and without Staphylococcus
aureus infection. Hairless SKH-1 inbred mice (6-8 weeks of age)
were anesthetized with ether and a midline 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 fascia. Incisions were closed using steel clips placed
at 1 cm intervals.
[0092] Formulations of neutral soluble glucan in phosphate buffered
saline were applied 30 minutes following wounding and reapplied at
24 hour intervals during the seven day post-operative period. Two
micrograms of neutral soluble glucan/mouse per day was topically
applied. Wounds were examined daily and rank-ordered for
effectiveness of formulation for enhancement of visual based wound
healing. Wounds were scored for closure on a scale of 0-5, with 5
indicating the most healing. In one group of mice infected, the
wound was treated with a culture of 10.sup.7 Staphylococcus aureus
30 minutes after wounding and 2 hrs prior to treatment with the
neutral soluble glucan formulation.
[0093] Histological evaluation of the wound site of each test group
was made. The dermis of the control group (untreated wound) was
heavily infiltrated with both lymphocytes and
monocytes/macrophages. However, re-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.
[0094] 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.
[0095] 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
Stimulation of Platelet Proliferation by Neutral Soluble Glucan
[0096] 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.
[0097] 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 peripheral blood counts. As shown in FIG.
7, the platelet cell count from neutral soluble glucan-treated mice
was increased nearly 3-fold relative to saline-treated control
levels.
[0098] 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 before injecting either saline or the neutral soluble glucan,
prepared as described in Example 1, intramuscularly in a single
dose of 0 (saline) or 2 mg/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 FIG. 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.
[0099] Biological Deposit
[0100] Saccharomyces cerevisiae strain R4 Ad was deposited on Aug.
20, 1992 with the American Type Culture Collection (ATCC), 12301
Parklawn Drive, Rockville, Md., 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.
[0101] Equivalents
[0102] 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.
* * * * *