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| United States Patent | 5,177,001 |
| Yamamoto | January 5, 1993 |
A novel macrophage activating factor is prepared in vitro by treating glycosylated mammalian vitamin D-binding protein with glycosidases. Vitamin D-binding protein, which is isolated from blood or plasma of animals by known procedures, is thus readily converted to a highly potent macrophage activating factor.
| Inventors: | Yamamoto; Nobuto (1040 66th Ave., Philadelphia, PA 19126) |
| Assignee: | Yamamoto; Nobuto (Philadelphia, PA) |
| Appl. No.: | 767742 |
| Filed: | September 30, 1991 |
| U.S. Class: | 435/68.1; 514/8; 530/380; 530/395; 530/402 |
| Intern'l Class: | C12P 021/02; A61K 037/04; C07K 003/08; C07K 009/00 |
| Field of Search: | 435/68.1 514/8,2 530/350,351,380,395,402,829 424/85.1 |
Yamamoto et al., Cancer Res. 47:2008, 1987. Yamamoto et al., Cancer Immunol. Immunother. 25:185, 1987. Yamamoto et al., Cancer Res. 42:6044, 1988. Ngwenya et al., Abstracts of the Annual Meeting of the American Society of Microbiology, Abs. E-72, p. 121 (1988). Homma, Abstracts of the Annual Meeting of the Am. Society of Microbiology, Abs. E-74, p. 121, (1988). Cooke et al., J. Clin. Invest. 76:2420-2424 (1985). Yang et al., Proc. Natl. Acad. Sci. 82:7994-7998 (1985). Haddad et al., Biochem. J. 218: 805-810, 1984. Link et al., Analyt. Biochem. 157: 262-269, 1986. Cooke et al., Endocrine Reviews 10: 294-307, 1989. Ogata et al., Comp. Biochem. Physiol. 90 B; 193-199, 1988. Gahne et al., Anim. Blood Grps. Biochem. Genet. 9: 37-40, 1978. Van De Weghe et al., Comp. Biochem. Physiol. 73 B: 977-982, 1982. Van Baelen et al., J. Biol. Chem. 253: 6344-6345, Sep. 25, 1978. Svasti et al., J. Biol. Chem. 253: 4188-4194, Jun. 25, 1978. Shinomiya et al., J. Biochem. 92: 1163-1171, 1982. |
FIELD OF THE INVENTION
The invention relates to macrophage activation, in particular to the in
vitro enzymatic production of a potent macrophage activating factor.
BACKGROUND OF THE INVENTION
A. Inflammatory Response Results in Activation of Macrophages
Microbial infections of various tissues cause inflammation which results in
chemotaxis and activation of phagocytes. Inflamed tissues release
lysophospholipids due to activation of phospholipase A. Inflamed cancerous
tissues produce alkyl-lysophospholipids and alkylglycerols as well as
lysophospholipids, because cancerous cells contain alkylphospholipids and
monoalkyldiacylglyercols. These lysophospholipids and alkylglycerols,
degradation products of membranous lipids in the inflamed normal and
cancerous tissues, are potent macrophage activating agents (Yamamoto et al.,
Cancer Res. 7:2008, 1987; Yamamoto et al., Cancer Immunol. Immunother.
25:185, 1987; Yamamoto et al., Cancer Res. 24:6044, 1988).
Administration of lysophospholipids (5-20 .mu.g/mouse) and alkylglycerols
(10-100 ng/mouse) to mice activates macrophages to phagocytize
immunoglobulin G-coated sheep red blood cells. The macrophages phagocytize
the target red blood cells via their receptors recognizing the Fc portion of
the immunoglobulin G but not the C3b portion of the complement (Yamamoto et
al., Cancer Res. 47:2008, 1987).
In vitro treatment of mouse peritoneal macrophages alone with
lysophospholipids or alkylglycerols results in no enhanced ingestion
activity (Yamamoto et al., Cancer Res. 48:6044, 1988). However, incubation
of peritoneal cells (mixture of macrophages and B and T lymphocytes) with
lysophospholipids or alkylglycerols for 2-3 hours produces markedly enhanced
Fc-receptor-mediated phagocytic activity of macrophages (Yamamoto et al.,
Cancer Res. 47:2008, 1987; Yamamoto et al., Cancer Res. 48:6044, 1988).
Incubation of macrophages with lysophospholipid- or alkylglycerol-treated B
and T lymphocytes in a medium containing 10% fetal calf serum developed a
greatly enhanced phagocytic activity of macrophages (Yamamoto et al., Cancer
Res. 48:6044, 1988; Homma and Yamamoto, Clin. Exp. Immunol. 79:307, 1990).
Analysis of macrophage activating signal transmission among the nonadherent
(B and T) lymphocytes has revealed that lysophospholipid- or alkylglycerol-treated
B-cells can transmit a signalling factor to T-cells; in turn, the T-cells
modify the factor to yield a new factor, which is capable of the ultimate
activation of macrophages for ingestion capability (Yamamoto et al., Cancer
Res. 48:6044, 1988).
B. Vitamin D-Binding Protein
Vitamin D-binding protein, also known as DBP, is an evolutionary conserved
glycoprotein among animals (Cooke and Haddad, Endocrine Rev. 10:294 1989).
DBP from animals serologically cross-reacts with human DBP (Ogata et al.,
Comp. Bioch. Physiol. 90B:193, 1988). Animal DBP is a genetically
polymorphic plasma protein in some species and has a relative molecular
weight of about 52,000. It normally constitutes about 0.5% of the plasma
proteins in animals. The plasma concentration is generally about 260 .mu.g/ml.
Polymorphism of the human DBP, known as "group specific component"
or "Gc protein" is demonstrable by gel electrophoretic analysis,
which reveals two major phenotypes: Gc1 and Gc2 (Hirschfeld et al., Nature
185:931, 1960). The entire nucleotide coding sequences of the Gc1 and Gc2
genes, and the predicted amino acid sequences, have been reported (Cooke, et
al., J. Clin. Invest. 76:2420, 1985; Yang et al., Proc. Natl. Acad. Sci. USA
82:7994, 1985). Gc1 is further divided into Gc1f and Gc1s subtypes which
migrate electrophoretically as two bands, "fast" and
"slow", (Svasti et al., Biochem. 18:1611, 1979).
Coopenhaver et al., Arch. Biochem. Biophys. 226, 218-223 (1983) reported
that a post-translational glycosylation difference occurs at a threonine
residue, which appeared in a region of the protein having an amino acid
difference between Gc1 and Gc2.
Viau et al., Biochem. Biophys. Res. Commun. 117, 324-331 (1983), reported a
predicted structure for the O-glucosidically linked glycan of Gc1,
containing a linear arrangement of sialic acid, galactose and N-acetylgalactosamine
linked to a serine or threonine residue.
Polymorphism of mammalian DBP can be demonstrated by isoelectric focusing (Gahne
and Juneja, Anim. Blood Grps. Biochem. Genet. 9:37, 1978; Van de Weghe et
al., Comp. Biochem. Physiol. 73B:977, 1982; Ogata et al., Comp. Biochem.
Physiol. 90B:193, 1988).
The animal DBP may be purified by a variety of means, which have been
reported in the literature. For example, DBP may be purified by
25-hydroxyvitamin D.sub.3 -Sepharose.RTM. affinity chromatography from
plasma of various animal species (Link, et al., Anal. Biochem. 157:262,
1986). DBP can also be purified by actin-agarose affinity chromatography due
to its specific binding capacity to actin (Haddad et al., Biochem. J.
218:805, 1984).
Despite the characterization and intensive study of the human and animal
vitamin D-binding protein, and the existence of ready methods for their
purification, the conversion of these proteins to a potent macrophage
activity factor has not been demonstrated until the present invention.
SUMMARY OF THE INVENTION
A process for the production of a potent macrophage activating factor is
provided. Animal vitamin D-binding protein, which is an evolutionary
conserved animal protein which is serologically cross-reactive with
group-specific component in human serum, is a precursor of the macrophage
activating factor. Animal DBP is converted to the macrophage activating
factor by the action of glycosidases of B and T cells.
According to a process for preparing macrophage activating factor, animal
DBP is contacted in vitro (i) with .beta.-galactosidase, or (ii) with
.beta.-galactosidase in combination with sialidase, .alpha.-mannosidase or a
mixture thereof. A potent macrophage activating factor is obtained in large
quantities.
According to one embodiment of the invention, animal DBP, which is believed
to possess an oligosaccharide moiety which includes galactose and sialic
acid residues (hereinafter "DBPgs"), is contacted with .beta.-galactosidase
and sialidase to provide the macrophage activating factor. According to
another embodiment, DBP which is believed to possess an oligosaccharide
moiety which includes galactose and .alpha.-mannose residues (hereinafter
"DBPgm") is contacted with .beta.-galactosidase and .alpha.-mannosidase.
In yet another embodiment, DBP which is believed to possess an
oligosaccharide moiety which includes a galactose residue without sialic
acid or .alpha.-mannose (hereinafter "DBPg") is contacted with
.beta.-galactosidase alone to form the macrophage activating factor. Because
of DBP genetic polymorphism, the macrophage activating factor is preferably
prepared by contacting animal DBP with all three enzymes to obtain the
macrophage activating factor, particularly when DBP purified from pooled
plasma of different individuals is utilized.
The invention also relates to a macrophage activating factor prepared
according to the above process or any embodiment thereof, and compositions
comprising the macrophage activating factor in combination with a
pharmaceutically acceptable carrier, for veterinary use.
The invention further relates to a method for inducing macrophage activation
in an animal in need thereof by administering to such animal a macrophage
activating effective amount of the novel macrophage activating factor.
"Animal DBP" as used herein means the genetically polymorphic
animal (exclusive of human) glycoprotein, also known as "vitamin
D-binding protein", including all genetic variations thereof, such as
DBPg, DBPgs and DBPgm. The singular expression "DBP" is thus
understood to encompass all such variants, unless stated otherwise.
By "macrophage activation" is meant the stimulation of macrophages
to an increased level of phagocytic activity.
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DETAILED DESCRIPTION OF THE INVENTION
A serum factor, which has been identified as animal DBP, is converted to a
macrophage activating factor by the action of B and T cell glycosidases. DBP
exists as a polypeptide having attached thereto a specific oligosaccharide,
portions of which are readily removable by treatment with readily available
glycosidases. These glycosidases are equivalent to the functions of B and T
cells upon the DBP. Upon treatment with specific glycosidases, DBP is
unexpectedly converted to a highly potent macrophage activating factor.
Thus, efficient conversion of DBP to the macrophage activating factor is
achieved in vitro, in the absence of B- and T-cells. The novel macrophage
activating factor formed by the enzymatic treatment of DBP is substantially
pure and of such high potency that administration to a host of even a trace
amount (500 picogram/kg of body weight) results in greatly enhanced
phagocytic macrophage activity. Since the enzymatic generation of the novel
factor bypasses the functions of B- and T-cells in macrophage activation, it
has utility as a potent adjuvant for vaccination and as a post-infection
therapeutic agent for serious infectious diseases.
T-cell lymphokine macrophage activating factor, also known as
.gamma.-interferon, is generated by lymphokine-producing T-cells in small
amounts, or is obtained by genetic engineering. The novel macrophage
activating factor of the invention, on the other hand, may be readily
obtained from DBP which can be readily purified from the plasma of animal
blood according to known purification procedures.
The polymorphic DBP phenotypes are expressed inter alia as differences in
the oligosaccharide attached to the polypeptide portion of the DBP molecule.
The novel macrophage activating factor of the invention may be efficiently
produced from animal DBP by incubation with a combination of .beta.-galactosidase
and sialidase, or a combination of .beta.-galactosidase and .alpha.-mannosidase.
In some instances, treatment of DBP with .beta.-galactosidase alone
efficiently yields the macrophage activating factor. The in vitro conversion
of DBP to macrophage activating factor by the action of commercially
available enzymes is so efficient that an extremely high activity of
macrophage activating factor is obtained.
Due to its genetic polymorphism in many animal species, DBP is preferably
treated with all three enzymes, as an enzyme mixture. In particular, DBP
obtained from pooled blood from several individuals of the species may
contain more than one DBP type. Complete conversion of DBP to macrophage
activating factor may thus most expeditiously be achieved by treatment with
all three enzymes, as an enzyme mixture.
DBPg treated with .beta.-galactosidase alone efficiently activates
macrophages. Therefore, removal of galactose from DPBg results in the
formation of the macrophage activating factor. On the other hand, two
glycosidases are required to convert DBP from DBPgs and DBPgm animals.
Conversion of DBPgs to macrophage activity factor requires incubation with
the combination of .beta.-galactosidase and sialidase. DBPgm conversion
requires .beta.-galactosidase and .alpha.-mannosidase.
It is believed that animal DBP phenotypes and subtypes are characterized as
glycoproteins having the following oligosaccharide structures linked to an
amino acid residue of the protein portion of the molecule:
______________________________________
Representative
DBP Type
Oligosaccharide Animal Species
______________________________________
DBPgs
##STR1## monkey, bovine, sheep, goat, pig, horse
DBPgm
##STR2## bovine
DBPg GalGalNAc dog, cat, rat, mouse
______________________________________
The glycosidases utilized in the practice of the invention are well known
and commercially available. .beta.-Galactosidase, (.beta.-D-galactosidase
galactohydrolase, EC 3.2.1.23) is obtained from Escherichia coli. .beta.-Galactosidase
is available, for example, from Boehringer Mannheim Biochemicals,
Indianapolis, Ind., cat. no. 634395.
.alpha.-Mannosidase (.alpha.-D-mannoside mannohydrolase, EC 3.2.1.24) is
obtained from the jack bean (Canavalia ensiformis). It is available, for
example, from Boehringer Mannheim Biochemicals, cat. no. 269611.
Sialidase, also known as "neuraminidase" (acylneuraminyl hydrolase
EC 3.2.1.18), is obtained from Clostridium perfringens, Vibrio cholerae or
Arthrobacter ureafaciens. All three forms of sialidase are available from
Boehringer Mannheim Biochemicals, cat. nos. 107590, 1080725 and 269611.
DBP is readily converted to the macrophage activating factor by contact with
a hydrolytic-effective amount of one or more of the above glycosidases. Any
amount of enzyme sufficient to achieve substantially complete conversion of
DBP to macrophage activating factor may be utilized. About 0.1 units (1 unit
being the amount of enzyme which catalyzes 1 .mu.mole of substrate in 1
minute) of each enzyme per 1 .mu.g of DBP is more than sufficient for this
purpose. Preferably, an excess of the amount of enzyme actually necessary to
convert the glycoprotein to macrophage activating factor is utilized to
insure complete conversion.
The DBP and enzymes may be contacted in, for example, phosphate buffer or
acetate buffer. A phosphate buffer is preferred (pH 5.5). Other media known
to those skilled in the art for conducting enzymatic reactions may be
substituted.
The reaction may be carried out at any temperature suitable for conducting
enzymatic reactions. Typically, the temperature may vary from 25.degree. C.
to 37.degree. C., with about 37.degree. C. being preferred. The substrate
and enzyme(s) are allowed to incubate in the reaction media until
substantial conversion of DBP to macrophage activating factor is achieved.
While it may be appreciated that the actual incubation times employed may
depend upon several factors such as the concentration of the reactants, the
reaction temperature, and the like, a reaction time of about 30 minutes at
37.degree. C. is generally sufficient to obtain complete conversion of DBP
to macrophage activating factor.
Conversion of DBP to macrophage activating factor may be conducted in any
vessel suitable for enzymatic reactions. It is preferred that sialidase is
utilized in insoluble form, e.g., attached to beaded agarose (Sigma Chemical
Co., cat. no. N-4483), to avoid contamination of the resulting macrophage
activating factor with sialidase fragments of similar molecular weight. The
macrophage activating factor may be produced by adding the appropriate
enzyme(s) to DBP in a liquid medium, followed by subsequent filtration of
the liquid to recover the macrophage activating factor. For example, the
enzyme-DBP reaction mixture may be passed through a sterilized 100 kDa cut
off filter (e.g. Amicon YM 100) to remove the immobilized sialidase, .beta.-galactosidase
(MW=540 kDa) and .alpha.-mannosidase (MW=190 kDa). The filtrate contains
substantially pure macrophage activating factor of high activity. Where the
conversion of large quantities of DBP to macrophage activating factor is
desired, all enzymes are most advantageously contained in the solid phase.
.beta.-Galactosidase, and sialidase or .alpha.-mannosidase, most preferably
a mixture of all three enzymes, is affixed to, e.g., agarose beads with a
suitable coupling agent such as cyanogen bromide. Methods for attaching
enzymes to solid supports are known to those skilled in the art. Conversion
of DBP to macrophage activating factor by means of incubation with
immobilized enzymes is preferred, as the subsequent step of separating the
macrophage activating factor from the enzyme mixture is obviated.
Regardless of whether immobilized or liquid phase enzyme is utilized, it is
desired to pass the product mixture through an ultrafilter, preferably a
filter having a pore size no larger than about 0.45.mu., to provide an
aseptic preparation of macrophage activating factor.
B-cells possess the function corresponding to .beta.-galactosidase. T-cells
carry the functions corresponding to sialidase and .alpha.-mannosidase.
Without wishing to be bound by any theory, it is believed that DBP is
modified in vivo in an ordered sequence by the membranous enzymes of B and T
lymphocytes to yield macrophage activating factor.
Activation of macrophages, which is characterized by their consequent
enhanced phagocytic activity, is the first major step in a host's immune
defense mechanism. Macrophage activation requires B and T lymphocyte
functions, which modify DBP in a step-wise fashion, to yield the novel
macrophage activating factor. Since the glycosidases used for in vitro
conversion of DBP to macrophage activating factor according to the present
invention correspond to the B- and the T-cell function required for
production of macrophage activating factor, the in vitro enzymatic
generation of the macrophage activating factor bypasses the functions of B-
and T-cells. Moreover, since the herein described macrophage activating
factor may be generated from blood of the same animal species undergoing
treatment, side effects, such as immunogenicity, are believed to be minimal.
Following infection, microbial antigens are bound by macrophages. Most of
this surface-bound antigen is internalized (i.e., phagocytized), and
processed by digestion. The macrophages return some processed antigens to
their surfaces so that antigenic determinants can be "presented"
efficiently to antigen-specific lymphocytes. However, the binding,
phagocytosis, processing and presentation of antigens requires that the
macrophage first be activated. Development of the immune response following
infection is thus typically delayed for 1-2 weeks, pending complete
macrophage activation. This is the period during which B- and T-cells
participate in generating the macrophage activating factor. During this lag
period, the infection may become well-established.
I have observed the occurrence of macrophage activation in mice in less than
six hours following administration of the macrophage activating factor
prepared from DBP. Substantial antibody production is observed in mice in as
little as 48 hours after coinjection of the macrophage activating factor and
antigen. A large amount of antigen-specific antibody is produced within 96
hours. It is thus contemplated that the macrophage activating factor of the
present invention, which is capable of inducing extemely rapid activation of
macrophages, will be useful as an adjuvant for vaccination to enhance and
accelerate the development of the immune response and to generate a large
amount of antigen-specific antibodies. For the same reason, it is further
contemplated that the macrophage activating factor will find utility as a
post-infection therapeutic agent to accelerate antibody production, either
alone or in combination with other therapeutic agents. This therapy should
be particularly effective in treating infectious diseases with long
incubating periods, such as rabies.
To minimize any possible immunologic reaction from administration of the
macrophage activating factor, it is preferred that each animal species would
receive only macrophage activating factor derived from the blood of the same
species. Similarly, the risk of immunologic reaction in individual animals
would be minimized by administering only the same variant of DBP-derived
macrophage activating factor, in situations wherein there is intraspecies
DBP polymorphism.
The macrophage activating factor may be administered to an animal to induce
macrophage activation, either alone or in combination with other therapies.
The amount of macrophage activating factor administered depends on a variety
of factors, including the potency of the agent, the duration and degree of
macrophage activation sought, the size and weight of the subject, the nature
of the underlying affliction, and the like. Generally, administration of as
little as about 0.5 ng of factor per kg of the subject's body weight will
result in substantial macrophage activation. According to one treatment, an
animal may receive about 2 ng of macrophage activating factor per kilogram
of body weight every three to five days to maintain a significant level of
macrophage activation.
The macrophage activating factor may be administered by any convenient means
which will result in delivery to the circulation of an amount of the factor
sufficient to induce substantial macrophage activation. For example, it may
be delivered by intravenous or intramuscular injection. Intramuscular
administration is presently preferred as the route of administration.
The macrophage activating factor may be taken up in pharmaceutically
acceptable carriers, particularly those carriers suitable for delivery of
proteinaceous pharmaceuticals. The factor is soluble in water or saline
solution. Thus, the preferred formulation for veterinary pharmacological use
comprises a saline solution of the agent. The formulation may optionally
contain other agents, such as agents to maintain osmotic balance. For
example, a typical carrier for injection may comprise an aqueous solution of
0.9% NaCl or phosphate buffered saline (a 0.9% NaCl aqueous solution
containing 0.01M sodium phosphate, .apprxeq.pH 7.0).
The invention is illustrated by the following non-limiting examples.
EXAMPLE 1
A. Conversion of DBP to Macrophage Activating Factor
Purified DBP (1.0 .mu.g) obtained from (A) cow, (B) pooled blood of seven
cows, (C) cat or (D) dog was combined with 1 ml of phosphate-buffered saline
(PBS-Mg) containing 0.01M sodium phosphate, 0.9% NaCl and 1 mM MgSC.sub.4
and treated with 2 .mu.l of PBS-Mg containing 0.1 U of the enzyme
combinations indicated in Table 1. The enzymes utilized were as follows:
Sialidase (Boehringer Mannheim Biochemicals, cat. no. 107590).
.alpha.-Mannosidase (Boehringer, cat. no. 107379).
.beta.-Galactosidase (Boehringer, cat. no. 634395).
The respective enzyme-DBP mixtures were incubated in microcentrifuge tubes
for sixty minutes at 37.degree. C. The reaction mixture containing the
enzyme-treated DBP was then diluted 10.sup.-4 in 0.1% egg albumin (EA)
supplemented medium, for the following assay.
B. In Vitro Assay of Macrophage Activating-Factor
1. Preparation of Macrophage Tissue Culture
Peritoneal cells were collected by injecting 5 ml of phosphate buffered
saline, containing 0.01M sodium phosphate, 0.9% NaCl and 5 units/ml heparin
into the peritoneal cavity of BALB/c mice. Peritoneal cells were removed and
washed by low speed centrifugation and suspended in a tissue culture medium
RPMI 1640 supplemented with 0.1% egg albumin (EA medium) at a concentration
of 1-2.times.10.sup.6 cells/ml. 1 ml aliquots of the cell suspension were
layered onto 12 mm coverglasses which had been placed in the 16 mm diameter
wells of tissue culture plates (Costar, Cambridge, Mass.). The plates were
incubated at 37.degree. C. in a 5% CO.sub.2 incubator for 30 minutes to
allow macrophage adherence to the coverglass. The coverglasses were removed,
immersed with gentle agitation in RPMI medium to dislodge non-adherent B and
T cells, and placed in fresh tissue culture wells containing EA-medium.
2. Preparation of Sheep Erythrocyte/Rabbit Anti-erythrocyte IgG Conjugates
Washed sheep erythrocytes were coated with subagglutinating dilutions of the
purified IgG fraction of rabbit anti-sheep erythrocyte antibodies. A 0.5%
suspension of rabbit IgG-coated sheep erythrocytes in RPMI 1640 medium was
prepared for use in the following phagocytosis assay.
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3. Phagocytosis Assay.
1 ml aliquots of the diluted reaction mixture from A., above, were layered
onto the macrophage-coated cover-glasses from B.1., above, and incubated for
2 hours in a 5% CO.sub.2 incubator at 37.degree. C. The culture media was
then removed and 0.5 ml of the 0.5% erythrocyte-IgG conjugate suspension
were added to the macrophage-coated cover-glasses and incubated for 1 hour
at 37.degree. C. The coverglasses were then washed in a hypotonic solution
(1/5 diluted phosphate buffered saline in water) to lyse non-ingested
erythrocytes. The macrophages with ingested erythrocytes were counted. The
average number of erythrocytes ingested per macrophage was also determined.
Macrophage phagocytic activity was calculated as an "Ingestion
index" (the percentage of macrophages which ingested erythrocytes times
the average number of erythrocytes ingested per macrophage). The data are
set forth in Table 1.
TABLE 1
______________________________________
Macrophage Activation by
Glycosidase-treated DBP
Ingestion Index
B
Glycosidases for
A .sup. pooled.sup.
C D
treatment of DBP
bovine.sup.
bovine.sup.
cat dog
______________________________________
-- 55 .+-. 10
67 .+-. 15
77 .+-. 12
73 .+-. 19
Sialidase 59 .+-. 15
71 .+-. 19
80 .+-. 21
59 .+-. 10
.beta.-galactosidase
63 .+-. 18
76 .+-. 15
278 .+-. 35
284 .+-. 41
Mannosidase
61 .+-. 13
73 .+-. 28
69 .+-. 15
62 .+-. 26
.beta.-galactosidase +
295 .+-. 34
335 .+-. 32
269 .+-. 31
265 .+-. 37
sialidase
.alpha.-mannosidase +
67 .+-. 22
54 .+-. 12
73 .+-. 20
67 .+-. 26
sialidase
.beta.-galactosidase +
72 .+-. 15
188 .+-. 38
266 .+-. 38
252 .+-. 33
mannosidase
______________________________________
It is apparent from Table 1 that bovine species display polymorphism with
respect to DBP type. While the purified DBP from a single bovine individual
(column A) was converted to macrophage activating factor by treatment with a
combination of sialidase and .beta.-galactosidase, treatment with .beta.-galactosidase
and either sialidase or .alpha.-mannosidase resulted in generation of
macrophage activator from DBP purified from pooled bovine plasma of seven
cows. It is thus apparent that the single bovine individual was of DBP type
"gs" and that the pooled material was composed of DBP from both
DBPgs and DBPgm individuals. Similarly, it is apparent from Table 1 that the
cat and dog DBP donors were type DBPg, since treatment with galactosidase
alone was sufficient for generation of macrophage activating factor.
The effect of macrophage activating factor concentration on activity was
investigated by treating the same bovine DBPgs, pooled bovine DBP, and cat
DBPg according to Example 1, at glycosidase-treated DBP dilutions of
10.sup.-4, 10.sup.-5 and 10.sup.-6 of the original 1.0 .mu.g/ml solution.
The results are set forth in Table 2 (bovine DBPgs), Table 3 (pooled bovine
DBP) and Table 4 (cat DBPg).
TABLE 2
______________________________________
Macrophage activation by
Glycosidase-treated Bovine DBPgs
Ingestion Index
Dilution of Bovine DBP
Glycosidase- Bovine DBP treated with
Treated untreated .beta.-galactosidase
Bovine DBPgs control and sialidase
______________________________________
10.sup.-4 63 .+-. 12 289 .+-. 11
10.sup.-5 59 .+-. 15 322 .+-. 35
10.sup.-6 55 .+-. 18 116 .+-. 22
______________________________________
TABLE 3
______________________________________
Macrophage Activation by
Glycosidase-treated pooled bovine DBP
Dilution of
Ingestion Index
Glycosidase-
Bovine Bovine DBP Bovine DBP
Treated pooled
DBP treated with
treated with
Bovine DBPgs
untreated
.beta.-galactosidase
.beta.-galactosidase
and DBPgm control and sialidase
and .alpha.-mannosidase
______________________________________
10.sup.-4 72 .+-. 25
312 .+-. 38 285 .+-. 38
10.sup.-5 83 .+-. 20
297 .+-. 45 203 .+-. 36
10.sup.-6 76 .+-. 18
145 .+-. 34 122 .+-. 23
______________________________________
TABLE 4
______________________________________
Macrophage Activation by
Glycosidase-treated Cat DBPg
Ingestion Index
Dilution of Cat DBP
Glycosidase- untreated
Cat DBP treated with
Cat DBPg control .beta.-galactosidase
______________________________________
10.sup.-4 68 .+-. 26
320 .+-. 29
10.sup.-5 65 .+-. 23
275 .+-. 23
10.sup.-6 76 .+-. 20
108 .+-. 34
______________________________________
EXAMPLE 3
Purified DBP (1.0 .mu.g from each of the species identified in Table 5,
below, was treated according to Example 1 with a mixture of .beta.-galactosidase,
sialidase and .alpha.-manosidase (0.5 U each) in 1 ml of PBS-Mg containing
0.01M sodium phosphate, 0.9% NaCl and 1 mM MgSO.sub.4 for sixty minutes at
37.degree. C. The reaction mixture containing each treated DBP was then
diluted 10.sup.-4 in 0.1% supplemented EA medium and assayed for macrophage
activation activity according to the in vitro assay of Example 1B. The
results are set forth in Table 5. It may be observed that treatment with a
mixture containing all three enzymes resulted in conversion of DBP to a
potent macrophage activating factor, regardless of DBP polymorphism.
TABLE 5
______________________________________
Ingestion Index
Treated with
Glycosidase- Untreated .beta.-galactosidase +
mannosidase control sialidase + .alpha.
______________________________________
Monkey (Macaca fucata)
72 .+-. 26
295 .+-. 38
Bovine (Bos taurus)
52 .+-. 19
320 .+-. 52
Sheep (Ovis aries)
48 .+-. 17
313 .+-. 48
Goat (Capra hircus)
56 .+-. 24
289 .+-. 32
Pig (Sus scrofa)
47 .+-. 12
332 .+-. 27
Horse (Equus caballus)
69 .+-. 23
266 .+-. 38
Cat (Felis catus)
58 .+-. 15
328 .+-. 43
Dog (Canis familigris)
60 .+-. 17
337 .+-. 18
Rat (Fisher) 65 .+-. 25
284 .+-. 37
Mouse (BALB/C)
71 .+-. 28
276 .+-. 34
______________________________________
DBP in 1 ml of PBS-Mg (pH 5.5) was combined with a mixture of the
above-prepared agarose-immobilized enzymes (2 units each enzyme) in 1 ml of
PBS-Mg (pH 5.5). The reaction mixtures were incubated in 5 ml plastic tubes
at 37.degree. C. in an end-over-end mixer for 30 minutes. The reaction
mixtures were thereafter spun with a table-top centrifuge at 2,000 rpm for
15 minutes. The supernatant of each reaction mixture was collected, filtered
through a sterilized 0.45.mu. pore size filter (type HA, Millipore Company,
Bedford, Mass.), and diluted.
B. In Vivo Assay of Macrophage Activating Factor
The enzymatically-modified DBP (100, 30, 10, 3 and 1 picogram samples) were
administered intramuscularly to BALB/c mice weighing .about.20 grams. At 18
hours post-administration, peritoneal cells were collected and placed on 12
mm coverglasses in the 16 mm wells of tissue culture plates. The plates were
incubated at 37.degree. C. for 30 minutes to allow adherence of macrophages.
The coverglasses were washed in RPMI 1640 medium to dislodge non-adherent
cells, and then placed in new wells. Rabbit IgG-coated sheep erythrocytes as
prepared in Example 1B.2. were layered onto the coverglass, and a
phagocytosis assay was performed as in Example 1B.3. The results are set
forth in Table 6:
TABLE 6
______________________________________
In Vivo Assay of Macrophage Activation
by Glycosidase-treated Bovine DBPgs
Dosage of
Ingestion Index
enzymatically
Bovine DBP Dog DBP
modified treated with treated with
DBP .beta.-galacto- .beta.-galacto-
(picogram/
untreated
sidase and untreated
sidase and
mouse) control sialidase control
sialidase
______________________________________
100 63 .+-. 18
283 .+-. 42
55 .+-. 22
272 .+-. 29
30 56 .+-. 17
341 .+-. 38
43 .+-. 12
295 .+-. 35
10 52 .+-. 18
315 .+-. 44
63 .+-. 17
277 .+-. 41
3 51 .+-. 12
141 .+-. 27
51 .+-. 15
128 .+-. 27
1 65 .+-. 15
86 .+-. 12
60 .+-. 18
89 .+-. 26
______________________________________
| ( 1 of 1 ) |
| United States Patent | 5,177,002 |
| Yamamoto | January 5, 1993 |
A novel, potent macrophage activating factor is prepared in vitro by treating glycosylated human group-specific component, also known as human vitamin D-binding protein, with glycosidases. Group-specific component, which is isolated from retired blood by known procedures, is thus readily converted to a highly potent macrophage activating factor.
| Inventors: | Yamamoto; Nobuto (1040 66th Ave., Philadelphia, PA 19126) |
| Assignee: | Yamamoto; Nobuto (Philadelphia, PA) |
| Appl. No.: | 576248 |
| Filed: | August 31, 1990 |
| U.S. Class: | 435/68.1; 514/8; 530/380; 530/395; 530/402 |
| Intern'l Class: | C12P 021/02; A61K 037/04; C07K 003/08; C07K 009/00 |
| Field of Search: | 435/68.1 514/8,2 530/350,351,380,402,829,395 424/85.1 |
J. Svasti et al., Journal of Biological Chemistry 253:4188-4194, Jun. 25, 1978. H. Van Baelen et al., Journal of Biological Chemistry 253:6344-6345, Sep. 25, 1978. Cooke et al., J. Clin. Invest. 76:2420, 1985. Yang et al., Proc. Natl. Acad. Sci. 82:7994, 1985. Yamamoto et al., Cancer Res. 47:2008, 1987. Yamamoto et al., Cancer. Immunol. Immunother. 25:185, 1987. Yamamoto et al., Cancer Res. 24:6044, 1988. Ngwenya et al., Abstracts of the Annual Meeting of the American Society of Microbiology, Abstract E-72, p. 121 (1988). Homma, Abstracts of the Annual Meeting of the American Society of Microbiology, Abstract E-74, p. 121 (1988). |
Viau et al., Biochem. Biophys. Res. Commun. 117, 324-331 (1983),
reported a predicted structure for the O-glucosidically linked glycan of
Gc1, containing a linear arrangement of sialic acid, galactose and N-acetylgalactosamine
linked to a serine or threonine residue.
The Gc protein may be purified by a variety of means, which have been
reported in the literature. For example, the Gc protein may be purified
by 25-hydroxy-vitamin D.sub.3 -Sepharose.RTM. affinity chromatography
from retired blood of the American Red Cross (Link, et al., Anal.
Biochem. 157:262, 1986). The Gc protein can also be purified by
actin-agarose affinity chromatography due to its specific binding
capacity to actin (Haddad et al., Bioch J. 218:805, 1984).
Despite the characterization and intensive study of the human vitamin
D-binding protein, and the existence of ready methods for its
purification, the enzymatic conversion of this protein to a potent
macrophage activity factor has not been demonstrated until the present
invention.
SUMMARY OF THE INVENTION
A process for the production of a potent macrophage activating factor is
provided. Human vitamin D-binding protein, which is identical to
group-specific component in human serum, is a precursor of the
macrophage activating factor. Group-specific component is converted to
the factor by the action of glycosidases of B and T cells.
According to a process for preparing macrophage activating factor,
group-specific component is contacted in vitro (i) with .beta.-galactosidase,
or (ii) with .beta.-galactosidase in combination with sialidase,
.alpha.-mannosidase or a mixture thereof. A potent macrophage activating
factor is obtained in large quantities
According to one embodiment of the invention, group-specific component
of phenotype Gc1, subtype Gc1f, is contacted with .beta.-galactosidase
and sialidase to provide the macrophage activating factor. According to
another embodiment, group-specific component of phenotype Gc1, subtype
Gc1s, is contacted with .beta.-galactosidase and .alpha.-mannosidase.
Preferably, group-specific component of phenotype Gc1, subtype Gc1s, is
contacted with not only .beta.-galactosidase and .alpha.-mannosidase,
but also sialidase, to ensure the conversion of the Gc1s variant
(hereinafter Gc1s*) which contains sialic acid in lieu of
.alpha.-mannose. Gc1s*, like Gc1f, requires treatment with .beta.-galactosidase
and sialidase for conversion to macrophage activating factor. In yet
another embodiment, group-specific component of phenotype Gc2 is
contacted with .beta.-galactosidase alone to form the macrophage
activating factor. Preferably, the macrophage activating factor is
prepared by contacting pooled group-specific components comprising a
mixture of Gc1f, Gc1s (Gc1s*) and Gc2 with all three enzymes to obtain
the macrophage activating factor.
The invention also relates to a macrophage activating factor prepared
according to the above process or any embodiment thereof, and
compositions comprising the macrophage activating factor in combination
with a pharmaceutically acceptable carrier.
The invention further relates to a method for inducing macrophage
activation in an individual in need thereof by administering to such an
individual macrophage activating effective amount of the novel
macrophage activating factor.
"Group-specific component" or "Gc protein" as used
herein means the genetically polymorphic glycoprotein, also known as
"vitamin D-binding protein", including all genetic variations
thereof, such as Gc2, Gc1, and subtypes such as Gc1f, Gc1s and Gc1s*.
The singular expression "group-specific component" or "Gc
protein" is thus understood to encompass all such variants, unless
stated otherwise.
By "macrophage activation" is meant the stimulation of
macrophages to an increased level of phagocytic activity.
DESCRIPTION OF THE FIGURES
FIGS. 1A-1B, and 2A-2B contain the reported amino acid sequence of human
group-specific component, phenotypes Gc1 and Gc2, respectively. The
underlined amino acid residues at positions 152, 311, 416 and 420 differ
between the two proteins.
DETAILED DESCRIPTION OF THE INVENTION
A serum factor, which has been identified as human group-specific
component, is converted to a macrophage activating factor by the action
of B and T cell glycosidases. Human group-specific component exists as a
polypeptide having attached thereto specific oligosaccharide moieties,
certain of which are readily removable by treatment with readily
available glycosidases. These glycosidases are equivalent to the
functions of B and T cells upon the Gc protein. Upon treatment with
specific glycosidases, group-specific component is unexpectedly
converted to a highly potent macrophage activating factor. Thus,
efficient conversion of Gc protein to the macrophage activating factor
is achieved in vitro, in the absence of intact B- and T-cells. The novel
macrophage activating factor formed by the enzymatic treatment of Gc
protein is substantially pure and of such high potency that
administration to a host of even a trace amount (500 picogram/kg of body
weight) results in greatly enhanced phagocytic macrophage activity.
Since the enzymatic generation of the novel factor bypasses the
functions of B-and T-cells in macrophage activation, it has utility as a
therapeutic agent for inducing macrophage activation, particularly in
individuals afflicted with immunodeficient diseases, cancer or other
immunocompromising diseases characterized by impaired B- or T-cell
function.
T-cell lymphokine macrophage activating factor, also known as
.gamma.-interferon, is generated by lymphokine-producing T-cells in
small amounts, or is obtained by genetic engineering. The novel
macrophage activating factor of the invention, on the other hand, may be
readily obtained from Gc protein which may be purified from the plasma
of retired human blood in large volume, according to known purification
procedures.
The human Gc protein phenotypes Gc1 and Gc2, and the Gc1 subtypes Gc1f
and Gc1s, are expressed inter alia as differences in the
oligosaccharides attached to the polypeptide portion of the Gc molecule.
The novel macrophage activating factor of the invention may be
efficiently produced from Gc1f or Gc1s protein by incubation with a
combination of .beta.-galactosidase and sialidase, or a combination of
.beta.-galactosidase and .alpha.-mannosidase, respectively. If the Gc1s
comprises at least in part the Gc1s variant, Gc1s*, which contains
sialic acid (N-acetyl-D-neuramic acid, or "NeuNAc") in lieu of
.alpha.-mannose, the mixture of enzymes utilized to treat the Gc1s/Gc1s*
mixture advantageously also includes sialidase. Treatment of the Gc2
protein with .beta.-galactosidase alone efficiently yields the
macrophage activating factor. The in vitro conversion of Gc protein to
macrophage activating factor by the action of commercially available
enzymes is so efficient that an extremely high activity of macrophage
activating factor is obtained.
Due to its genetic polymorphism, Gc protein obtained from pooled retired
human blood will likely contain all three principal Gc types. Complete
conversion of a mixture of Gc proteins to macrophage activating factor
may thus most expeditiously be achieved by treatment with all three
enzymes, as an enzyme mixture.
The molecules of the Gc1 and the Gc2 phenotypes are believed to differ
by four amino acids at positions 152, 311, 416 and 420, as reported in
the literature and reproduced in FIGS. 1A-1B and 2A-2B. The differences
are as follows: ##STR1##
All three principal Gc types--Gc1s, Gc1f and Gc2--differ in the nature
of the appended oligosaccharide, although it is believed that most Gc2
molecules are unglyosylated. Only the glycosylated form of Gc2 is a
precursor for macrophage activating factor according to the process
described herein. Incubation of each of Gc1f, Gc1s and Gc2 molecules
with galactose-specific lectin beads absorbed all three macrophage
activator precursor types. Thus, the outer oligosaccharide moiety of
each of the three principal human Gc types is believed to be galactose.
Gc2 protein treated with .beta.-galactosidase alone efficiently
activates macrophages. Therefore, removal of galactose from Gc2 protein,
to the extent the molecule is present in its glycosylated form, results
in the formation of the macrophage activating factor. On the other hand,
two glycosidases are required to convert the Gc1 proteins to macrophage
activating factor. Conversion of Gc1f to the macrophage activity factor
requires incubation with the combination of .beta.-galactosidase and
sialidase. Conversion of Gc1s requires .beta.-galactosidase and .alpha.-mannosidase
(or .beta.-galactosidase and sialidase, in the case of Gc1s*).
The innermost sugar of the oligosaccharide moiety of Gc1 protein is N-acetylgalactosamine
(Coppenhaver et al., Arch Biochem. Biophys. 226, 218-223, 1983).
Treatment of Gc1 protein with endo-N-acetylglucosaminidase, which
results in the cleavage of the N-acetylgalactosamine, results in a
molecule which cannot be then converted to macrophage activating factor.
It is believed that the Gc protein phenotypes and subtypes are
characterized as glycoproteins having the following oligosaccharide
structures linked to an amino acid residue of the protein portion of the
molecule: ##STR2##
Without wishing to be bound by any theory, it is believed that the above
glycosylation occurs at the specific protein portion of the Gc
glycoprotein through a threonine residue occurring at amino acid
position 420 (Gc1 phenotype) or through a threonine residue occurring at
neighboring amino acid position 418 (Gc2 phenotype), thus forming the O-glycosidic
linkage GalNAc.alpha.(1.fwdarw.0)-Thr. Thus, without wishing to be bound
by any theory, the novel macrophage activating factor is believed to
comprise a protein in substantially pure form having substantially the
amino acid sequence of human group specific component, and a terminal N-acetylgalactosamine
group linked to an amino acid residue, most likely threonine 420 and/or
418.
Further without wishing to be bound by any theory, it is believed that
Gc types 1f and 1s* have the same oligosaccharide moiety, but differ in
amino acid sequence. The site of the yet unknown variation in amino acid
sequence is postulated to be present in the vicinity of amino acid
position 420.
Human Gc protein of high purity for use in the process of the invention
is most readily prepared by 25-hydroxyvitamin D.sub.3 -Sepharose.RTM.
affinity chromatography from retired blood according to the procedure of
Link et al., Anal. Buiochem. 157, 262 (1986), the entire disclosure of
which is incorporated herein by reference. The Gc protein may also be
purified by actin-agarose affinity chromatography according to the
procedure of Haddad et al., Biochem. J. 218, 805 (1984), which takes
advantage of the binding specificity of Gc protein for actin. The entire
disclosure of Haddad et al., is incorporated herein by reference. Other
methods of obtaining Gc protein in high purity are reported in the
literature.
The glycosidases utilized in the practice of the invention are well
known and commercially available. Galactosidase, (.beta.-D-galactosidase
galactohydrolase, Ec 3.2.1.23) is obtained from Escherichia coli.
.beta.-Galactosidase is available, for example, from Boehringer Mannheim
Biochemicals, Indianapolis, Ind., cat. no. 634395.
.alpha.-Mannosidase (.alpha.-D-mannoside mannohydrolase, EC 3.2.1.24) is
obtained from the jack bean (Canavalia ensiformis). It is available, for
example, from Boehringer Mannheim Biochemicals, cat. no. 269611.
Sialidase, also known as "neuraminidase" (acylneuraminyl
hydrolase EC 3.2.1.18), is obtained from Clostridium perfringens, Vibrio
cholerae or Arthrobacter ureafaciens. All three forms of sialidase are
available from Boehringer Mannheim Biochemicals, cat. nos. 107590,
1080725 and 269611.
Gc protein is readily converted to the macrophage activating factor by
contact with a hydrolytic-effective amount of one or more of the above
glycosidases. Any amount of enzyme sufficient to achieve substantially
complete conversion of Gc protein to macrophage activating factor may be
utilized. About 0.1 units (1 unit being the amount of enzyme which
catalyzes 1 .mu.mole of substrate in 1 minute) of each enzyme per 2.6 .mu.g
of Gc protein is more than sufficient for this purpose. Preferably, an
excess of the amount of enzyme actually necessary to convert the
glycoprotein to macrophage activating factor is utilized to insure
complete conversion.
The Gc protein and enzymes may be contacted in, for example, phosphate
buffer or acetate buffer. A phosphate buffer is preferred (pH 5.5).
Other media known to those skilled in the art for conducting enzymatic
reactions may be substituted.
The reaction may be carried out at any temperature suitable for
conducting enzymatic reactions. Typically, the temperature may vary from
25.degree. C. to 37.degree. C., with about 37.degree. C. being
preferred. The substrate and enzyme(s) are allowed to incubate in the
reaction media until substantial conversion of the Gc protein to
macrophage activating factor is achieved. While it may be appreciated
that the actual incubation times employed may depend upon several
factors such as the concentration of the reactants, the reaction
temperature, and the like, a reaction time of about 30 minutes at
37.degree. C. is generally sufficient to obtain complete conversion of
Gc protein to macrophage activating factor.
Conversion of Gc protein to macrophage activating factor may be
conducted in any vessel suitable for enzymatic reactions. It is
preferred that sialidase is utilized in insoluble form, e.g., attached
to beaded agarose (Sigma Chemical Co., cat. no. N-4483), to avoid
contamination of the resulting macrophage activating factor with
sialidase fragments of similar molecular weight. The macrophage
activating factor may be produced by adding the appropriate enzyme(s) to
Gc protein in a liquid medium, followed by subsequent filtration of the
liquid to recover the macrophage activating factor. For example, the
enzyme-Gc protein reaction mixture may be passed through a sterilized
100 kDa cut off filter (e.g. Amicon YM 100) to remove the immobilized
sialidase, .beta.-galactosidase (MW=540 kDa) and .alpha.-mannosidase
(MW=190 kDa). The filtrate contains substantially pure macrophage
activating factor of high activity.
Where the conversion of large quantities of Gc protein to macrophage
activating factor is desired, all enzymes are most advantageously
contained in the solid phase. .beta.-Galactosidase, and sialidase or
.alpha.-mannosidase, most preferably a mixture of all three enzymes, is
affixed to, e.g., agarose beads with a suitable coupling agent such as
cyanogen bromide. Methods for attaching enzymes to solid supports are
known to those skilled in the art. Conversion of Gc protein to
macrophage activating factor by means of incubation with immobilized
enzymes is preferred, as the subsequent step of separating the
macrophage activating factor from the enzyme mixture is obviated.
Regardless of whether immobilized or liquid phase enzyme is utilized, it
is desired to pass the product mixture through an ultrafilter,
preferably a filter having a pore size no larger than about 0.45.mu., to
provide an aseptic preparation of macrophage activating factor.
Without wishing to be bound by any theory, it is believed that B-cells
possess the function corresponding to .beta.-galactosidase, and that
T-cells carry the functions corresponding to sialidase and .alpha.-mannosidase.
It is believed that Gc protein is modified in vivo in an ordered
sequence by the membranous enzymes of B and T lymphocytes to yield
macrophage activating factor.
Activation of macrophages, which is characterized by their consequent
enhanced phagocytic activity, is the first major step in a host's immune
defense mechanism. Macrophage activation requires B and T lymphocyte
functions, which modify Gc protein in a step-wise fashion, to yield the
novel macrophage activating factor. Since the glycosidases used for in
vitro conversion of Gc protein to macrophage activating factor according
to the present invention correspond to the B- and the T-cell function
required for production of macrophage activating factor, the in vitro
enzymatic generation of the macrophage activating factor bypasses the
functions of B- and T-cells. Thus, in vitro enzymatic-generated
macrophage activating factor may be used for the therapy of immuno-deficient
diseases, cancer and other disease conditions characterized by the
immunocompromise of the afflicted individual. Moreover, since the herein
described in vitro-generated macrophage activating factor is of human
origin, side effects, such as immunogenicity, are believed to be
minimal.
To minimize any possible immunologic reaction from administration of the
macrophage activating factor, it is preferred that individuals of
phenotype Gc1 would receive only Gc1 -derived macrophage activating
factor. Similarly, the risk of immunologic reaction in Gc2 individuals
would be minimized by administering only Gc2-derived macrophage
activating factor.
The novel macrophage activating factor is also believed useful in the
treatment of disorders characterized by a disruption or loss of B- or
T-cell function. Such disorders may be characterized by a lack of
macrophage activation. Addition of exogenous macrophage activating
factor of the invention will result in the restoration of macrophage
activity, even in the absence of complete B- or T-cell function.
The macrophage activating factor may be administered to an individual to
induce macrophage activation, either alone or in combination with other
therapies. The amount of macrophage activating factor administered
depends on a variety of factors, including the potency of the agent, the
duration and degree of macrophage activation sought, the size and weight
of the subject, the nature of the underlying affliction, and the like.
Generally, administration of as little as about 0.5 ng of factor per kg
of the subject's body weight will result in substantial macrophage
activation. According to one treatment, a human subject may receive as
little as about 30-35 ng of macrophage activating factor every three to
five days to maintain a significant level of macrophage activation.
The macrophage activating factor may be administered by any convenient
means which will result in delivery to the circulation of an amount of
the factor sufficient to induce substantial macrophage activation. For
example, it may be delivered by intravenous or intramuscular injection.
Intravenous administration is presently preferred as the route of
administration.
The macrophage activating factor may be taken up in pharmaceutically
acceptable carriers, particularly those carriers suitable for delivery
of proteinaceous pharmaceuticals. The factor is soluble in water or
saline solution. Thus, the preferred formulation for pharmacological use
comprises a saline solution of the agent. The formulation may optionally
contain other agents, such as adjuvants to maintain osmotic balance. For
example, a typical carrier for injection may comprise an aqueous
solution of 0.9% NaCl or phosphate buffered saline (a 0.9% NaCl aqueous
solution containing 0.01M sodium phosphate, .apprxeq.pH 7.0).
The invention is illustrated by the following non-limiting examples.
EXAMPLE 1
A. Conversion of Gc Protein to Macrophage Activating Factor
Gc protein (2.6 .mu.g; Gc1 or Gc2) in 1 ml of phosphate-buffered saline
(PBS-Mg) containing 0.01M sodium phosphate, 0.9% NaCl and 1 mM
MgSO.sub.4 was treated with 2 .mu.l of PBS-Mg containing 0.1 U of the
following enzymes or enzyme combinations.
Gc1f/Gc1s* conversion: sialidase (Boehringer Mannheim Biochemicals, cat.
no. 107590) and .beta.-galactosidase (Boehringer, cat. no. 634395);
Gc1s conversion: .alpha.-mannosidase (Boehringer, cat. no. 107379) and
.beta.-galactosidase;
Gc2 conversion: .beta.-galactosidase only.
The respective enzyme-Gc protein mixtures were incubated in
microcentrifuge tubes for thirty minutes at 37.degree. C. The reaction
mixture containing the treated Gc protein was then diluted 10.sup.-4,
10.sup.-5 or 10.sup.-6 in 0.1% egg albumin (EA) medium, for the
following assay.
B. In Vitro Assay of Macrophage Activating Factor
1. Preparation of Macrophage Tissue Culture
Peritoneal cells were collected by injecting 5 ml of phosphate buffered
saline, containing 0.01M sodium phosphate, 0.9% NaCl and 5 units/ml
heparin into the peritoneal cavity of BALB/c mice. Peritoneal cells were
removed and washed by low speed centrifugation and suspended in a tissue
culture medium RPMI 1640 supplemented with 0.1% egg albumin (EA) medium
at a concentration of 1-2.times.10.sup.6 cells/ ml. 1 ml aliquots of the
cell suspension were layered onto 12 mm coverglasses which had been
placed in the 16 mm diameter wells of tissue culture plates (Costar,
Cambridge, Mass.). The plates were incubated at 37.degree. C. in a 5%
CO.sub.2 incubator for 30 minutes to allow macrophage adherence to the
coverglass. The coverglasses were removed, immersed with gentle
agitation in RPMI medium to dislodge non-adherent B and T cells, and
placed in fresh tissue culture wells containing EA-medium.
2. Preparation of Sheet Erythrocyte/Rabbit Anti-erythrocyte IgG
Conjugates
Washed sheep erythrocytes were coated with subagglutinating dilutions of
the purified IgG fraction of rabbit anti-sheep erythrocyte antibodies. A
0.5% suspension of rabbit IgG-coated sheep erythrocytes in RPMI 1640
medium was prepared for use in the following phagocytosis assay.
3. Phagocytosis Assay
1 ml aliquots of the diluted reaction mixture from A., above, were
layered onto the macrophage-coated coverglasses from B.1., above, and
incubated for 2 hours in a 5% CO.sub.2 incubator at 37.degree. C. The
culture media was then removed and 0.5 ml of the 0.5% erythrocyte-IgG
conjugate suspension were added to the macrophage-coated coverglasses
and incubated for 1 hour at 37.degree. C. The coverglasses were then
washed in a hypotonic solution (1/5 diluted phosphate buffered saline in
water) to lyse non-ingested erythrocytes. The macrophages with ingested
erythrocytes were counted. The average number of erythrocytes ingested
per macrophage was also determined. Macrophage phagocytic activity was
calculated as an "Ingestion index" (the percentage of
macrophages which ingested erythrocytes.times.the average number of
erythrocytes ingested per macrophage). The data is set forth in Table 1
(Gc1 ) and Table 2 (Gc2).
TABLE 1
______________________________________
Dilution of
Ingestion Index
Glycosidase-
Gc1 Gc1 treated with
Gc1 treated with
Treated untreated
.beta.-galactosidase
.beta.-galactosidase
Gc1.sup.1 Protein
control and sialidase
and .alpha.-mannosidase
______________________________________
10.sup.-4
75 .+-. 10
352 .+-. 15 295 .+-. 11
10.sup.-5
82 .+-. 11
286 .+-. 11 210 .+-. 8
10.sup.-6
79 .+-. 8
122 .+-. 7 109 .+-. 13
______________________________________
.sup.1 Mixture of Gc1f and Gc1s
TABLE 2
______________________________________
Dilution of Ingestion Index
Glycosidase- Gc2
Treated untreated
Gc2 treated with
Gc2 Protein control .beta.-galactosidase
______________________________________
10.sup.-4 65 .+-. 13
325 .+-. 16
10.sup.-5 69 .+-. 11
208 .+-. 17
10.sup.-6 71 .+-. 20
116 .+-. 5
______________________________________
2. Conversion of Gc Protein to Macrophage Activating Factor
Gc protein (2.6 .mu.g; Gc1, Gc2, or mixture thereof) in 1 ml of PBS-Mg
(pH 5.5) was combined with a mixture of the above-prepared agarose-immobilized
enzymes (2 units each enzyme) in 1 ml of PBS-Mg (pH 5.5). The reaction
mixtures were incubated in 5 ml plastic tubes at 37.degree. C. in an
end-over-end mixer for 30 minutes. The reaction mixtures were thereafter
spun with a table-top centrifuge at 2,000 rpm for 15 minutes. The
supernatant of each reaction mixture was collected, filtered through a
sterilized 0.45.mu. pore size filter (type HA, Millipore Company,
Bedford, Mass.), and diluted.
B. In Vivo Assay of Macrophage Activating Factor
The enzymatically-modified Gc protein (40, 10, 4 and 1 picogram samples)
were administered intramuscularly to BALB/c mice weighing .about.20
grams. At 18 hours post-administration, peritoneal cells were collected
and placed on 12 mm coverglasses in the 16 mm wells of tissue culture
plates. The plates were incubated at 37.degree. C. for 30 minutes to
allow adherence of macrophages. The coverglasses were washed in RPMI
1640 medium to dislodge non-adherent cells, and then placed in new
wells. Rabbit IgG-coated sheep erythrocytes as prepared in Example 1B.2.
were layered onto the coverglass, and a phagocytosis assay was performed
as in Example 1B.3. The results are set forth in Table 3:
TABLE 3
______________________________________
Dosage of enzyma-
Ingestion Index
tically modified
Untreated Control
Glycosidase-treated
Gc protein Gc1 + Gc1 +
(picogram/mouse)
Gc1 Gc2 Gc1 Gc2
______________________________________
40 57 .+-. 16
59 .+-. 7
322 .+-. 19
314 .+-. 11
10 55 .+-. 10
63 .+-. 13
353 .+-. 16
332 .+-. 14
4 51 .+-. 12
45 .+-. 8
163 .+-. 18
152 .+-. 13
1 63 .+-. 18
56 .+-. 9
114 .+-. 3
106 .+-. 5
______________________________________
| ( 1 of 1 ) |
| United States Patent | 5,177,004 |
| Schutt | January 5, 1993 |
The present invention relates to an improved process for the preparation of 1-desoxynojirimycin. 1-Desoxynojirimycin can be reacted by alkylation on the nitrogen atom to give various saccharase inhibitors which are used therapeutically in the treatment of diabetes mellitus.
| Inventors: | Schutt; Hermann (Wuppertal, DE) |
| Assignee: | Bayer Aktiengesellschaft (Leverkusen, DE) |
| Appl. No.: | 757867 |
| Filed: | September 11, 1991 |
| U.S. Class: | 435/84; 435/85; 435/228; 435/229; 435/230 |
| Intern'l Class: | C12P 019/26; C12P 019/28; C12N 009/80; C07H 005/06 |
| Field of Search: | 435/84,85,230,228,229 |
| 3880713 | Apr., 1975 | Fleming et al. | 435/230. |
| 3930949 | Jan., 1976 | Kutzbach et al. | 435/230. |
| 3962036 | Jun., 1976 | Liersch et al. | 435/230. |
| 4264734 | Apr., 1981 | Kahan et al. | 435/228. |
| 4282322 | Aug., 1981 | Kahan et al. | 435/228. |
| 4405714 | Sep., 1983 | Kinast et al. | 435/84. |
| 4806650 | Feb., 1989 | Schroder et al. | 435/84. |
| 4981789 | Jan., 1991 | Lein | 435/228. |
| Foreign Patent Documents | |||
| 0049858 | Apr., 1982 | EP. | |
| 0240868 | Oct., 1987 | EP. | |
The present invention relates to an improved process for the
preparation of 1-desoxynojirimycin. 1-Desoxynojirimycin can be reacted
by alkylation on the nitrogen atom to give various saccharase inhibitors
which are used therapeutically in the treatment of diabetes mellitus (W.
Puls, U. Keup, H.P. Krause, G. Thomas, and F. Hoffmeister,
Naturwissenschaften, 64 (1977) 536 et seq.).
1-Desoxynojirimycin is prepared by a combined chemical-microbiological
process (EP 49,858).
An important intermediate in the preparation of 1-desoxynojirimycin is
aminosorbose, which, however, is unstable. To remove the N-acetyl and N-formyl
protecting group, either extremely acidic or extremely alkaline
conditions have to be selected. Above pH 2 and below pH 13, virtually no
removal of the acyl group takes place. Under the abovementioned pH
conditions, however, considerable desoxyno3irimycin losses are observed.
These result on the one hand from cyclisation reactions of the
aminosorbose itself to give, for example, hydroxylated pyridines, on the
other hand, coloured secondary components which have not been specified
in more detail are formed. Thus, on acidic removal of the acyl group
only a yield of about 27 % of desoxynojirimycin was observed and on
alkaline removal a yield of about 60 % of desoxynojirimycin was
observed.
SUMMARY OF THE INVENTION
Surprisingly, it has now been found that in the range from 8 to 9.5, the
removal of an N-phenacetyl protecting group by means of the known enzyme
penicillin acylase (penicillin amidohydrolase (EC 3.5.1.11)) isolated
from Escherichia coli can be achieved with substantially better yield
and purity of 1-desoxynojirimycin than with chemical methods.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph comparing the yield of 1-desoxynojirinycin over time
when prepared by chemical methods and enzymatic methods.
DETAILED DESCRIPTION OF THE INVENTION
The reaction mentioned is described by the following reaction equations:
##STR1##
At pH 13, in contrast to pH 8-9, a very rapid decomposition of
aminosorbose is observed, which leads to the loss of 40-50 % of
1-desoxynojirimycin.
In the enzymatic removal of the N-phenacetyl protecting group using
penicillin acylase, however, this rapid decomposition is lacking, so
that the yield of 1-desoxynojirimycin can be increased. The yield can be
still further increased by an increase in the enzyme concentration and
thus a reduction of the cleavage time (FIG. 1).
Penicillin acylase has hitherto only been described for the removal of
the N-phenacetyl group on penicillin and cephalosporin parent
substances, amino acids and peptides (J.G. Shewale, B.S. Deshphande, V.K.
Sudhakaran and S.S. Ambedkar, Process Biochemistry, June 1990, 97-103).
The removal of the phenacetyl group from amino sugars using penicillin
acylase is new and also utilisable for other unstable N-acyl-aminopentoses
and aminohexoses.
In the process described, purified free and carrier-bound penicillin
acylase, for example, can be employed.
EXAMPLE 1
Use of soluble penicillin acylase
3 kg of N-phenacetyl-aminosorbitol are dissolved in 150 litres of water
and the pH of the solution is adjusted to about 4.5 and kept at this pH.
The solution is warmed to 37.degree. C. and about 6 kg of centrifuged
bacterial cells (Gluconobacter suboxydans) are added and the suspension
is aerated intensively with air whilst stirring for 4 hours.
In this period, the N-phenacetyl-aminosorbitol is quantitatively
oxidised to N-phenacetyl-aminosorbose microbiologically. The
Gluconobacter cells are completely removed from the suspension by
centrifugation, the clear supernatant is adjusted to pH 9 using sodium
hydroxide solution and 2.7 million units of penicillin acylase are added
(isolation of the enzyme and definition of the unit are described in the
literature reference: C. Kutzbach and E. Rauenbusch, Hoppe-Seyler's Z.
Physiol.-Chem., 354 (1974) 45-53). The removal of the phenylacetic acid
is monitored by means of high pressure liquid chromatography via the
determination of the liberated phenylacetic acid and/or the decrease in
the N-phenacetylaminosorbose. After a reaction time of about 45-60
minutes, the removal of protecting groups is complete and 270 g of
sodium borohydride are added. Reduction and cyclisation to give
1-desoxynojirimycin last 60 minutes. The excess of sodium borohydride is
then destroyed by addition of acetone or HC1.
Further working-up to give pure 1-desoxynojirimycin is carried out by
chromatography via ion exchangers and subsequent crystallisation as
described in EP 49,859 and in the literature reference: Y. Eze, S. Maruo,
K. Miyazaki and M. Kawamata, Agric. Biol. Chem., 49 (1985) 1119-1125.
After crystallisation, about 1.3 kg of 1-desoxynojirimycin (about 80 %
molar yield) having a purity by HPLC >95 % are obtained.
EXAMPLE 2
Use of carrier-bound penicillin acylase Carrying-out takes place as
described in Example 1. To remove the N-phenacetyl group, 5.0 million
units of penicillin acylase resin prepared according to DOS (German
Offenlegungsschrift) 2,215,687 are added. Before the isolation of
1-desoxynojirimycin, the enzyme resin is filtered off and washed.
The yield of 1-desoxynojirimycin is 1.4 kg (about 83 % molar yield)
having a purity of HPLC>95%.
______________________________________
High pressure liquid chromatography
______________________________________
Column:
LiChrospher RP 18.5 .mu.m
(Merck, Darmstadt);
250 mm .times. 4 mm
Eluent:
A: 0.05 M phosphate, pH 6.5
B:90% acetonitrile: 10% water
Gradient:
Time in min. % A % B
0 100 0
2 100 0
10 0 100
11.5 0 100
11.6 100 0
16.5 100 0
Flow rate: 1.5 ml/min.
Temperature: 25.degree. C.
Wavelength: 210 nm
Injection volume: 20 .mu.L
Retention times in minutes:
N-phenacetyl-aminosorbitol:
7.1
N-phenacetyl-aminosorbose:
8.7
Phenylacetic acid:
4.7
Desoxynojirimycin:
0.9
______________________________________
| ( 1 of 1 ) |
| United States Patent | 5,326,749 |
| Yamamoto | * July 5, 1994 |
A novel macrophage activating factor is prepared in vitro by treating glycosated vitamin D-binding protein with glycosidases. Vitamin D-binding protein, which is isolated from blood or plasma of mammals by known procedures, is thus readily converted to a highly potent macrophage activating factor.
| Inventors: | Yamamoto; Nobuto (1040 66th Ave., Philadelphia, PA 19126) |
| [*] Notice: | The portion of the term of this patent subsequent to August 31, 2010 has been disclaimed. |
| Appl. No.: | 000320 |
| Filed: | January 4, 1993 |
| U.S. Class: | 514/8; 530/362; 530/380; 530/395 |
| Intern'l Class: | A61K 037/02; C07K 007/10; C07K 009/00; C07K 015/14 |
| Field of Search: | 514/8 530/380,395,362 |
| 5177001 | Jan., 1993 | Yamamoto | 435/68. |
| 5177002 | Jan., 1993 | Yamamoto | 435/68. |
Yamamoto et al., Cancer Research 47:2008, 1987. Yamamoto et al., Cancer Immunol. Immunother. 25:185, 1987. Yamamoto et al., Cancer Res. 48:6044, 1988. Ngwenya et al., Abstracts of the Annual Meeting of the American Society of Microbiology, Abs. E-72, p. 121 (1988). Homma, Abstracts of the Annual Meeting of the Am. Society of Microbiology, Abs. E-74, p. 121 (1988). Cooke et al., J. Clin. Invest. 76:2420-2424 (1985). Yang et al., Proc. Natl. Acad. Sci. 82:7994-7998 (1985). Biological Abstracts 68(5) p. 2831 (1979) Abs. No. 28360. Yamamoto, et al., Proc. Natl. Acad. Sci. USA 88, 8539-8543 (1991). Haddad et al., Biochem. J. 218:805, 1984. Link et al., Analyt. Biochem. 157: 262-269, 1986. Cooke et al., Endocrine Reviews 10:294-307, 1989. Ogata et al., Comp. Biochem. Physiol. 90B:193-199, 1988. Van De Weghe et al., Comp. Biochem. Physiol. 73B977-982, 1982. Van Baelen et al. J. Biol. Chem. 253:6344-6345, Sep. 25, 1978. Svasti et al., J. Biol. Chem. 253:4188-4194, Jun. 25, 1978. Shinomiya et al., J. Biochem. 92:1163-1171, 1982. Gahne et al., Anim. Blood Groups Biochem. Genet. 9, 37-40 (1978). Homma and Yamamoto, Clin. Exp. Immunol. 79, 307-313 (1990). Homma et al., Immunol. Cell. Biol. 68, 137-142 (1990). Yamamoto et al., Immunology 74, 420-424 (1991). Yamamoto et al., J. Immunol. 147, 273-280 (Jul. 1, 1991). Viau et al., "Isolation and characterization of the O-glycan chain of the human vitamin-D binding protein", Bch. B Phys. Res. Comm., 117:324-331 (1983). Coppenhaver et al., "Post-Translational Heterogeneity of the Human Vitamin D-Binding Protein", Arch. Biochem. Biophys. 226(1):218-223 (1983). Schulz et al., Principles of Protein Structure, pp. 14-16 (1979). |
Coopenhaver et al., Arch. Biochem. Biophys. 226, 218-223 (1983)
reported that a post-translational glycosylation difference occurs
at a threonine residue, which appeared in a region of the protein
having an amino acid difference between Gc1 and Gc2.
Viau et al.. Biochem. Biophys. Res. Commun. 117, 324-331 (1983),
reported a predicted structure for the O-glucosidically linked
glycan of Gc1, containing a linear arrangement of sialic acid,
galactose and N-acetylgalactosamine linked to a serine or threonine
residue. Polymorphism of mammalian DBP can be demonstrated by
isoelectric focusing (Gahne and Juneja, Anim. Blood Grps. Biochem.
Genet. 9:37, 1978; Van de Weghe et al., Comp. Biochem. Physiol.
73B:977, 1982; Ogata et al.. Comp. Biochem. Physiol. 90B:193, 1988).
DBP may be purified by a variety of means, which have been reported
in the literature. For example, DBP may be purified by
25-hydroxyvitamin D.sub.3 -Sepharose.RTM. affinity chromatography
from plasma of various animal species (Link, et al., Anal. Biochem.
157:262, 1986). DBP can also be purified by actinagarose affinity
chromatography due to its specific binding capacity to actin (Haddad
et al., Biochem. J. 218:805, 1984).
Despite the characterization and intensive study of the vitamin
D-binding protein, and the existence of ready methods for its
purification, the conversion of this protein to a potent macrophage
activity factor has not been demonstrated until the present
invention.
SUMMARY OF THE INVENTION
A process for the production of a potent macrophage activating
factor is provided. Vitamin D-binding protein, which is an
evolutionary conserved protein, is a precursor of the macrophage
activating factor. DBP is converted to the macrophage activating
factor by the action of glycosidases of B and T cells.
According to a process for preparing macrophage activating factor,
DBP is contacted in vitro (i) with .beta.-galactosidase, or (ii)
with .beta.-galactosidase in combination with sialidase, .alpha.-mannosidase
or a mixture thereof. A potent macrophage activating factor is
obtained in large quantities.
According to one embodiment of the invention, DBP, which possesses
an oligosaccharide moiety which includes galactose and sialic acid
residues (hereinafter "DBPgs"), is contacted with .beta.-galactosidase
and sialidase to provide the macrophage activating factor. According
to another embodiment, DBP which is believed to possess an
oligosaccharide moiety which includes galactose and .alpha.-mannose
residues (hereinafter "DBPgm") is contacted with .beta.-galactosidase
and .alpha.-mannosidase. In yet another embodiment, DBP which is
believed to possess an oligosaccharide moiety which includes a
galactose residue without sialic acid or .alpha.-mannose
(hereinafter "DBPg") is contacted with .beta.-galactosidase
alone to form the macrophage activating factor. Because of DBP
genetic polymorphism, the macrophage activating factor is preferably
prepared by contacting DBP with all three enzymes to obtain the
macrophage activating factor, particularly when DBP purified from
pooled plasma of different individuals is utilized.
The invention also relates to a macrophage activating factor
which may be prepared according to the above process or any
embodiment thereof, and compositions comprising the macrophage
activating factor in combination with a pharmaceutically acceptable
carrier, for pharmaceutical or veterinary use.
The invention further relates to a method for inducing macrophage
activation in a mammal in need thereof by administering to such
mammal a macrophage activating effective amount of the novel
macrophage activating factor.
"DBP" as used herein means the genetically polymorphic
vitamin D-binding protein also known as "group specific
component" ("Gc") in humans, including all genetic
variations thereof, such as DBPg, DBPgs and DBPgm. The singular
expression "DBP" is thus understood to encompass all such
variants, unless stated otherwise.
By "macrophage activation" is meant the stimulation of
macrophages to an increased level of phagocytic activity.
BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1A and 1B contain the reported amino acid sequence of DBPgs/gm.
The underlined amino acid residues at positions 152, 311, 416 and
420 differ from DBPg.
FIGS. 2A and 2B contain the reported amino acid sequence of DBPg.
The underlined amino acid residues at positions 152, 311, 416 and
420 differ from DBPgs/gm.
DETAILED DESCRIPTION OF THE INVENTION
A serum factor, which has been identified as DBP, is converted to a
macrophage activating factor by the action of B and T cell
glycosidases. DBP exists as a polypeptide having attached thereto a
specific oligosaccharide, portions of which are readily removable by
treatment with readily available glycosidases. These glycosidases
are equivalent to the functions of B and T cells upon the DBP. Upon
treatment with specific glycosidases, DBP is unexpectedly converted
to a highly potent macrophage activating factor. Thus, efficient
conversion of DBP to the macrophage activating factor is achieved in
vitro, in the absence of B- and T-cells. The novel macrophage
activating factor formed by the enzymatic treatment of DBP is
substantially pure and of such high potency that administration to a
host of even a trace amount (500 picogram/kg of body weight) results
in greatly enhanced phagocytic macrophage activity. Since the
enzymatic generation of the novel factor bypasses the functions of
B- and T-cells in macrophage activation, it has utility as a potent
adjuvant for vaccination and as a post-infection therapeutic agent
for serious infectious diseases.
T-cell lymphokine macrophage activating factor, also known as
.gamma.-interferon, is generated by lymphokine-producing T-cells in
small amounts, or is obtained by genetic engineering. The novel
macrophage activating factor of the invention, on the other hand,
may be readily obtained from DBP which can be readily purified from
the plasma of blood according to known purification procedures.
The polymorphic DBP phenotypes are expressed inter alia as
differences in the oligosaccharide attached to the polypeptide
portion of the DBP molecule. The novel macrophage activating factor
of the invention may be efficiently produced from DBP by incubation
with a combination of .beta.-galactosidase and sialidase, or a
combination of .beta.-galactosidase and .alpha.-mannosidase. In some
instances, treatment of DBP with .beta.-galactosidase alone
efficiently yields the macrophage activating factor. The in vitro
conversion of DBP to macrophage activating factor by the action of
commercially available enzymes is so efficient that an extremely
high activity of macrophage activating factor is obtained.
Due to its genetic polymorphism in many mammalian species, DBP is
preferably treated with all three enzymes, as an enzyme mixture. In
particular, DBP obtained from pooled blood from several individuals
of the species may contain more than one DBP type. Complete
conversion of DBP to macrophage activating factor may thus most
expeditiously be achieved by treatment with all three enzymes, as an
enzyme mixture.
The molecules of the DBPgs and DBPgm penotypes on the one hand (Gc1
in humans) is believed to differ from the DBPg phenotype (Gc2 in
humans) by four amino acids at positions 152, 311, 416 and 420, as
reported in the literature and reproduced in FIGS. 1 and 2. The
differences are as follows:
______________________________________
152 311 416 420
______________________________________
DBPgs, DBPgm (Gc1)
Glu Arg Glu Thr
DBPg (Gc2) Gly Glu Asp Lys
______________________________________
All three principal DBP types--DBPgm, DBPGs and DBPg--differ in
the nature of the appended oligosaccharide, although it is believed
that most DBP molecules having the amino acid sequence of DBPg
molecules are unglycosylated. Only the glycosylated form of DBPg is
a precursor for macrophage activating factor according to the
process described herein. Incubation of each of DBPgm, DBPgs and
DBPg molecules with galactose-specific lectin beads absorbed all
three macrophage activator precursor types. Thus, the outer
oligosaccharide moiety of each of the three principal human Gc types
is galactose.
DBPg treated with .beta.-galactosidase alone efficiently activates
macrophages. Therefore, removal of galactose from DPBg results in
the formation of the macrophage activating factor. On the other
hand, two glycosidases are required to convert DBP from DBPgs and
DBPgm individuals. Conversion of DBPgs to macrophage activity factor
requires incubation with the combination of .beta.-galactosidase and
sialidase. DBPgm conversion requires .beta.-galactosidase and
.alpha.-mannosidase.
The innermost sugar of the oligosaccharide moiety of DBPgs and DBPgm
protein is N-acetylgalactosamine (Coppenhaver et al., Arch. Biochem.
Biophys. 226, 218-223, 1983). Treatment of these glycoproteins with
endo-N-acetylglucosaminidase, which results in the cleavage of the
N-acetylgalactosamine, results in a molecule which cannot be then
converted to macrophage activating factor.
It is believed that DBP phenotypes and subtypes are characterized as
glycoproteins having the following oligosaccharide structures linked
to an amino acid residue of the protein portion of the molecule:
______________________________________
Representative
DBP Type
Oligosaccharide Species
______________________________________
DBPgs
##STR1## human (Gc1f, Gc1s*), monkey, bovine, sheep, goat,
pig, horse
DBPgm
##STR2## human (Gc1s, Gc1f*), bovine
DBPg GalGalNAc human (Gc2), dog, cat,
rat, mouse
______________________________________
Sialidase, also known as "neuraminidase" (acylneuraminyl
hydrolase EC 3.2.1.18), is obtained from Clostridium perfringens.
Vibrio cholerae or Arthrobacter ureafaciens. All three forms of
sialidase are available from Boehringer Mannheim Biochemicals, cat.
nos. 107590, 1080725 and 269611.
DBP is readily converted to the macrophage activating factor by
contact with a hydrolytic-effective amount of one or more of the
above glycosidases. Any amount of enzyme sufficient to achieve
substantially complete conversion of DBP to macrophage activating
factor may be utilized. About 0.1 units (1 unit being the amount of
enzyme which catalyzes 1 .mu.mole of substrate in 1 minute) of each
enzyme per 1 .mu.g of DBP is more than sufficient for this purpose.
Preferably, an excess of the amount of enzyme actually necessary to
convert the glycoprotein to macrophage activating factor is utilized
to insure complete conversion.
The DBP and enzymes may be contacted in, for example, phosphate
buffer or acetate buffer. A phosphate buffer is preferred (pH 5.5).
Other media known to those skilled in the art for conducting
enzymatic reactions may be substituted.
The reaction may be carried out at any temperature suitable for
conducting enzymatic reactions. Typically, the temperature may vary
from 25.degree. C. to 37.degree. C., with about 37.degree. C. being
preferred. The substrate and enzyme(s) are allowed to incubate in
the reaction media until substantial conversion of DBP to macrophage
activating factor is achieved. While it may be appreciated that the
actual incubation times employed may depend upon several factors
such as the concentration of the reactants, the reaction
temperature, and the like, a reaction time of about 30 minutes at
37.degree. C. is generally sufficient to obtain complete conversion
of DBP to macrophage activating factor.
Conversion of DBP to macrophage activating factor may be conducted
in any vessel suitable for enzymatic reactions. It is preferred that
sialidase is utilized in insoluble form, e.g., attached to beaded
agarose (Sigma Chemical Co., cat. no. N-4483), to avoid
contamination of the resulting macrophage activating factor with
sialidase fragments of similar molecular weight. The macrophage
activating factor may be produced by adding the appropriate enzyme(s)
to DBP in a liquid medium, followed by subsequent filtration of the
liquid to recover the macrophage activating factor. For example, the
enzyme-DBP reaction mixture may be passed through a sterilized 100
kDa cut off filter (e.g. Amicon YM 100) to remove the immobilized
sialidase, .beta.-galactosidase (MW=540 kDa) and .alpha.-mannosidase
(MW=190 kDa). The filtrate contains substantially pure macrophage
activating factor of high activity.
Where the conversion of large quantities of DBP to macrophage
activating factor is desired, all enzymes are most advantageously
contained in the solid phase. .beta.-Galactosidase, and sialidase or
.alpha.-mannosidase, most preferably a mixture of all three enzymes,
is affixed to, e.g., agarose beads with a suitable coupling agent
such as cyanogen bromide. Methods for attaching enzymes to solid
supports are known to those skilled in the art. Conversion of DBP to
macrophage activating factor by means of incubation with immobilized
enzymes is preferred, as the subsequent step of separating the
macrophage activating factor from the enzyme mixture is obviated.
Regardless of whether immobilized or liquid phase enzyme is
utilized, it is desired to pass the product mixture through an
ultrafilter, preferably a filter having a pore size no larger than
about 0.45.mu., to provide an aseptic preparation of macrophage
activating factor.
B-cells possess the function corresponding to .beta.-galactosidase.
T-cells carry the functions corresponding to sialidase and .alpha.-mannosidase.
Without wishing to be bound by any theory, it is believed that DBP
is modified in vivo in an ordered sequence by the membranous enzymes
of B and T lymphocytes to yield macrophage activating factor.
Activation of macrophages, which is characterized by their
consequent enhanced phagocytic activity, is the first major step in
a host's immune defense mechanism. Macrophage activation requires B
and T lymphocyte functions, which modify DBP in a step-wise fashion,
to yield the novel macrophage activating factor. Since the
glycosidases used for in vitro conversion of DBP to macrophage
activating factor according to the present invention correspond to
the B- and the T-cell function required for production of macrophage
activating factor, the in vitro enzymatic generation of the
macrophage activating factor bypasses the functions of B- and
T-cells. Moreover, since the herein described macrophage activating
factor may be generated from blood of the same mammalian species
undergoing treatment, side effects, such as immunogenicity, are
believed to be minimal.
Following infection, microbial antigens are bound by macrophages.
Most of this surface-bound antigen is internalized (i.e.,
phagocytized), and processed by digestion. The macrophages return
some processed antigens to their surfaces so that antigenic
determinants can be "presented" efficiently to
antigen-specific lymphocytes. However, the binding, phagocytosis,
processing and presentation of antigens requires that the macrophage
first be activated. Development of the immune response following
infection is thus typically delayed for 1-2 weeks, pending complete
macrophage activation. This is the period during which B- and
T-cells participate in generating the macrophage activating factor.
During this lag period, the infection may become well-established.
I have observed the occurrence of macrophage activation in mice in
less than six hours following administration of the macrophage
activating factor prepared from DBP. Substantial antibody production
is observed in mice in as little as 48 hours after coinjection of
the macrophage activating factor and antigen. A large amount of
antigen-specific antibody is produced within 96 hours. It is thus
contemplated that the macrophage activating factor of the present
invention, which is capable of inducing extemely rapid activation of
macrophages, will be useful as an adjuvant for vaccination to
enhance and accelerate the development of the immune response and to
generate a large amount of antigen-specific antibodies. For the same
reason, it is further contemplated that the macrophage activating
factor will find utility as a post-infection therapeutic agent to
accelerate antibody production, either alone or in combination with
other therapeutic agents. This therapy should be particularly
effective in treating infectious diseases with long incubating
periods, such as rabies.
To minimize any possible immunologic reaction from administration of
the macrophage activating factor, it is preferred that each
mammalian species would receive only macrophage activating factor
derived from the blood of the same species. Similarly, the risk of
immunologic in individuals would be minimized by administering only
the same variant of DBP-derived macrophage activating factor, in
situations wherein there is intraspecies DBP polymorphism.
The macrophage activating factor may be administered to an animal to
induce macrophage activation, either alone or in combination with
other therapies. The amount of macrophage activating factor
administered depends on a variety of factors, including the potency
of the agent, the duration and degree of macrophage activation
sought, the size and weight of the subject, the nature of the
underlying affliction, and the like. Generally, administration of as
little as about 0.5 ng of factor per kg of the subject's body weight
will result in substantial macrophage activation. According to one
treatment, a mammal may receive about 2 ng of macrophage activating
factor per kilogram of body weight every three to five days to
maintain a significant level of macrophage activation.
The macrophage activating factor may be administered by any
convenient means which will result in delivery to the circulation of
an amount of the factor sufficient to induce substantial macrophage
activation. For example, it may be delivered by intravenous or
intramuscular injection. Intramuscular administration is presently
preferred as the route of administration.
The macrophage activating factor may be taken up in pharmaceutically
acceptable carriers, particularly those carriers suitable for
delivery of proteinaceous pharmaceuticals. The factor is soluble in
water or saline solution. Thus, the preferred formulation for
veterinary pharmacological use comprises a saline solution of the
agent. The formulation may optionally contain other agents, such as
agents to maintain osmotic balance. For example, a typical carrier
for injection may comprise an aqueous solution of 0.9% NaCl or
phosphate buffered saline (a 0.9% NaCl aqueous solution containing
0.01M sodium phosphate, .apprxeq.pH 7.0).
The invention is illustrated by the following non-limiting examples.
EXAMPLE 1
A. Conversion of Human DBP (Gc protein) to Macrophage Activating
Factor
Gc protein (2.6 .mu.g; Gc1 or Gc2) in 1 ml of phosphate-buffered
saline (PBS-Mg) containing 0.01M sodium phosphate, 0.9% NaCl and 1
mM MgSO.sub.4 was treated with 2 .mu.l of PBS-Mg containing 0.1 U of
the following enzymes or enzyme combinations.
Gclf/Gcls* conversion: sialidase (Boehringer Mannheim Biochemicals,
cat. no. 107590) and .beta.-galactosidase (Boehringer, cat. no.
634395);
Gcls conversion: .alpha.-mannosidase (Boehringer, cat. no. 107379)
and .beta.-galactosidase;
Gc2 conversion: .beta.-galactosidase only.
The respective enzyme-Gc protein mixtures were incubated in
microcentrifuge tubes for thirty minutes at 37.degree. C. The
reaction mixture containing the treated Gc protein was then diluted
10.sup.-4, 10.sup.-5 or 10.sup.-6 in 0.1% egg albumin (EA) medium,
for the following assay.
B. In Vitro Assay of Macrophage Activating Factor
1. Preparation of Macrophage Tissue Culture
Peritoneal cells were collected by injecting ml of phosphate
buffered saline, containing 0.01M sodium phosphate, 0.9% NaCl and 5
units/ml heparin into the peritoneal cavity of BALB/c mice.
Peritoneal cells were removed and washed by low speed centrifugation
and suspended in a tissue culture medium RPMI 1640 supplemented with
0.1% egg albumin (EA) medium at a concentration of
1-2.times.10.sup.6 cells/ ml. 1 ml aliquots of the cell suspension
were layered onto 12 mm coverglasses which had been placed in the 16
mm diameter wells of tissue culture plates (Costar, Cambridge,
Mass.). The plates were incubated at 37.degree. C. in a 5% CO.sub.2
incubator for 30 minutes to allow macrophage adherence to the
coverglass. The coverglasses were removed, immersed with gentle
agitation in RPMI medium to dislodge non-adherent B and T cells, and
placed in fresh tissue culture wells containing EA-medium. 2.
Preparation of Sheep Erythrocyte/Rabbit Anti-erythrocyte IgG
Conjugates
Washed sheep erythrocytes were coated with subagglutinating
dilutions of the purified IgG fraction of rabbit anti-sheep
erythrocyte antibodies. A 0.5% suspension of rabbit IgG-coated sheep
erythrocytes in RPMI 1640 medium was prepared for use in the
following phagocytosis assay.
3. Phagocytosis Assay
1 ml aliquots of the diluted reaction mixture from A., above, were
layered onto the macrophage-coated coverglasses from B.1., above,
and incubated for 2 hours in a 5% CO.sub.2 incubator at 37.degree.
c. The culture media was then removed and 0.5 ml of the 0.5%
erythrocyte-IgG conjugate suspension were added to the
macrophage-coated coverglasses and incubated for 1 hour at
37.degree. C. The coverglasses were then washed in a hypotonic
solution (1/5 diluted phosphate buffered saline in water) to lyse
non-ingested erythrocytes. The macrophages with ingested
erythrocytes were counted. The average number of erythrocytes
ingested per macrophage was also determined. Macrophage phagocytic
activity was calculated as an "Ingestion index"(the
percentage of macrophages which ingested erythrocytes .times. the
average number of erythrocytes ingested per macrophage). The data is
set forth in Table 1 (Gc1) and Table 2 (Gc2).
TABLE 1
______________________________________
Dilution of Ingestion Index
Glycosidase-
Gc1 Gc1 treated with
Gc1 treated with
Treated untreated
.beta.-galactosidase
.beta.-galactosidase
Gc1.sup.1 Protein
control and sialidase
and .alpha.-mannosidase
______________________________________
10.sup.-4
75 .+-. 10
352 .+-. 15 295 .+-. 11
10.sup.-5
82 .+-. 11
286 .+-. 11 210 .+-. 8
10.sup.-6
79 .+-. 8
122 .+-. 7 109 .+-. 13
______________________________________
.sup.1 Mixture of Gc1f and Gc1s
TABLE 2
______________________________________
Dilution of Ingestion Index
Glycosidase- Gc2
Treated Untreated
Gc2 treated with
Gc2 Protein control .beta.-galactosidase
______________________________________
10.sup.-4 65 .+-. 13
325 .+-. 16
10.sup.-5 69 .+-. 11
208 .+-. 17
10.sup.-6 71 .+-. 20
116 .+-. 5
______________________________________
EXAMPLE 2
Conversion of Human DB (Gc Protein) to Macrophage Activating Factor
with Immobilized Enzyme
1. Preparation of Immobilized Enzymes
100 mg of CNBr-activated agarose (Sepharose.RTM. 4 B) was washed
with 1 mM HCl and suspended in coupling buffer (300 .mu.l)
containing NaHCO.sub.3 buffer (0.1M, pH 8.3) and NaCl (0.5M).
.beta.-Galactosidase, .alpha.-mannosidase and sialidase (2 U each
enzyme) were mixed in 600 .mu.l of the coupling buffer and incubated
at room temperature for 2 hours in an end-over-end mixer. Remaining
active groups in the agarose were blocked by incubation with 0.2M
glycine in coupling buffer for 2 hours at room temperature. The
agarose-immobilized enzyme was washed with coupling buffer to remove
unabsorbed protein and glycine, followed by washing with acetate
buffer (0.1M, pH 4) containing NaCl (0.5M), and additional coupling
buffer. The agarose-immobilized enzyme preparations were stored at
4.degree. C.
2. Conversion of Gc protein to Macrophage Activating Factor
Gc protein (2.6 .mu.g; Gc1, Gc2, or mixture thereof) in 1 ml of
PBS-Mg (pH 5.5) was combined with a mixture of the above-prepared
agarose-immobilized enzymes (2 units each enzyme) in 1 ml of PBS-Mg
(pH 5.5). The reaction mixtures were incubated in 5 ml plastic tubes
at 37.degree. C. in an end-over-end mixer for 30 minutes. The
reaction mixtures were thereafter spun with a table-top centrifuge
at 2,000 rpm for 15 minutes The supernatant of each reaction mixture
was collected, filtered through a sterilized 0.45.mu. pore size
filter (type HA, Millipore Company, Bedford, Mass.), and diluted.
B. In Vivo Assay of Macrophage Activating Factor
The enzymatically-modified Gc protein (40, 10, 4 and 1 picogram
samples) were administered intramuscularly to BALB/c mice weighing
.about.20 grams. At 18 hours post-administration, peritoneal cells
were collected and placed on 12 mm coverglasses in the 16 mm wells
of tissue culture plates. The plates were incubated at 37.degree. C.
for 30 minutes to allow adherence of macrophages. The coverglasses
were washed in RPMI 1640 medium to dislodge non-adherent cells, and
then placed in new wells. Rabbit IgG-coated sheep erythrocytes as
prepared in Example 1B.2. were layered onto the coverglass, and a
phagocytosis assay was performed as in Example 1B.3. The results are
set forth in Table 3:
TABLE 3
______________________________________
Dosage of
enzymatically
modified Ingestion Index
Gc protein
Untreated Glycosidase-
(picogram/
Control treated
mouse) Gc1 Gc1 + Gc2 Gc1 Gc1 + Gc2
______________________________________
40 57 .+-. 16
59 .+-. 7 322 .+-. 19
314 .+-. 11
10 55 .+-. 10
63 .+-. 13
353 .+-. 16
332 .+-. 14
4 51 .+-. 12
45 .+-. 8 163 .+-. 18
152 .+-. 13
1 63 .+-. 18
56 .+-. 9 114 .+-. 3
106 .+-. 5
______________________________________
EXAMPLES 3
A. Conversion of Animal DBP to Macrophage Activating Factor
Purified DBP (1.0 .mu.g) obtained from (A) cow, (B) pooled blood of
seven cows, (C) cat or (D) dog was combined with 1 ml of
phosphate-buffered saline (PBS-Mg) containing 0.01M sodium
phosphate, 0.9% NaCl and 1 mM MgSO.sub.4 and treated with 2 .mu.l of
PBS-Mg containing 0.1 U of the enzyme combinations indicated in
Table 4. The enzymes utilized were as follows:
Sialidase (Boehringer Mannheim Biochemicals, cat. no. 107590).
.alpha.-Mannosidase (Boehringer, cat. no. 107379).
.beta.-Galactosidase (Boehringer, cat. no. 634395)
The respective enzyme-DBP mixtures were incubated in microcentrifuge
tubes for sixty minutes at 37.degree. C. The reaction mixture
containing the enzyme-treated DBP was then diluted 10.sup.-4 in 0.1%
egg albumin (EA) supplemented medium, for the following assay.
B. In Vitro Assay of Macrophage Activating-Factor
1. Preparation of Macrophage Tissue Culture
Peritoneal cells were collected by injecting 5 ml of phosphate
buffered saline, containing 0.01M sodium phosphate, 0.9% NaCl and 5
units/ml heparin into the peritoneal cavity of BALB/c mice.
Peritoneal cells were removed and washed by low speed centrifugation
and suspended in a tissue culture medium RPMI 1640 supplemented with
0.1% egg albumin (EA medium) at a concentration of
1-2.times.10.sup.6 cells/ ml. 1 ml aliquots of the cell suspension
were layered onto 12 mm coverglasses which had been placed in the 16
mm diameter wells of tissue culture plates (Costar, Cambridge,
Mass.). The plates were incubated at 37.degree. C. in a 5% CO.sub.2
incubator for 30 minutes to allow macrophage adherence to the
coverglass. The coverglasses were removed, immersed with gentle
agitation in RPMI medium to dislodge non-adherent B and T cells, and
placed in fresh tissue culture wells containing EA-medium.
2. Preparation of Sheep Erythrocyte/Rabbit Anti-erythrocyte IgG
Conjugates
Washed sheep erythrocytes were coated with subagglutinating
dilutions of the purified IgG fraction of rabbit anti-sheep
erythrocyte antibodies. A 0.5% suspension of rabbit IgG-coated sheep
erythrocytes in RPMI 1640 medium was prepared for use in the
following phagocytosis assay.
3. Phagocytosis Assay
1 ml aliquots of the diluted reaction mixture from A., above, were
layered onto the macrophage-coated coverglasses from B.1., above,
and incubated for 2 hours in a 5% CO.sub.2 incubator at 37.degree.
C. The culture media was then removed and 0.5 ml of the 0.5%
erythrocyte-IgG conjugate suspension were added to the
macrophage-coated cover-glasses and incubated for 1 hour at
37.degree. C. The coverglasses were then washed in a hypotonic
solution (1/5 diluted phosphate buffered saline in water) to lyse
non-ingested erythrocytes. The macrophages with ingested
erythrocytes were counted. The average number of erythrocytes
ingested per macrophage was also determined. Macrophage phagocytic
activity was calculated as an "Ingestion index" (the
percentage of macrophages which ingested erythrocytes times the
average number of erythrocytes ingested per macrophage). The data
are set forth in Table 4.
TABLE 4
______________________________________
Macrophage Activation by
Glycosidase-treated DBP
Ingestion Index
A B C D
Glycosidases for pooled
treatment of DBP
bovine bovine cat dog
______________________________________
-- 55 .+-. 10
67 .+-. 15
77 .+-. 12
73 .+-. 19
Sialidase 59 .+-. 15
71 .+-. 19
80 .+-. 21
59 .+-. 10
.beta.-galactosidase
63 .+-. 18
76 .+-. 15
278 .+-. 35
284 .+-. 41
.alpha.-Mannosidase
61 .+-. 13
73 .+-. 28
69 .+-. 15
62 .+-. 26
.beta.-galactosidase +
295 .+-. 34
335 .+-. 32
269 .+-. 31
265 .+-. 37
sialidase
.alpha.-mannosidase +
67 .+-. 22
54 .+-. 12
73 .+-. 20
67 .+-. 26
sialidase
.beta.-galactosidase +
72 .+-. 15
188 .+-. 38
266 .+-. 38
252 .+-. 33
.alpha.-mannosidase
______________________________________
It is apparent from Table 4 that bovine species display
polymorphism with respect to DBP type. While the purified DBP from a
single bovine individual (column A) was converted to macrophage
activating factor by treatment with a combination of sialidase and
.beta.-galactosidase, treatment with .beta.-galactosidase and either
sialidase or .alpha.-mannosidase resulted in generation of
macrophage activator from DBP purified from pooled bovine plasma of
seven cows. It is thus apparent that the single bovine individual
was of DBP type "gs" and that the pooled material was
composed of DBP from both DBPgs and DBPgm individuals. Similarly, it
is apparent from Table 4 that the cat and dog DBP donors were type
DBPg, since treatment with galactosidase alone was sufficient for
generation of macrophage activating factor.
The effect of macrophage activating factor concentration on activity
was investigated by treating the same bovine DBPgs, pooled bovine
DBP, and cat DBPg according to Example 3, at glycosidase-treated DBP
dilutions of 10.sup.-4, 10.sup.-5 and 10.sup.-6 of the original 1.0
.mu.g/ml solution. The results are set forth in Table 5 (bovine
DBPgs), Table 6 (pooled bovine DBP) and Table 7 (cat DBPg).
TABLE 5
______________________________________
Macrophage activation by
Glycosidase-treated Bovine DBPgs
Ingestion Index
Dilution of Bovine DBP
Glycosidase- Bovine DBP treated with
Treated untreated .beta.-galactosidase
Bovine DBPgs control and sialidase
______________________________________
10.sup.-4 63 .+-. 12 289 .+-. 11
10.sup.-5 59 .+-. 15 322 .+-. 35
10.sup.-6 55 .+-. 18 116 .+-. 22
______________________________________
TABLE 6
______________________________________
Macrophage Activation by
Glycosidase-treated pooled bovine DBP
Ingestion Index
Dilution of Bovine DBP
Glycosidase- Bovine DBP treated with
Treated pooled
Bovine DBP treated with
.beta.-galactosidase
Bovine DBPgs
untreated .beta.-galactosidase
and .alpha.-
and DBPgm control and sialidase
mannosidase
______________________________________
10.sup.-4 72 .+-. 25 312 .+-. 38 285 .+-. 38
10.sup.-5 83 .+-. 20 297 .+-. 45 203 .+-. 36
10.sup.-6 76 .+-. 18 145 .+-. 34 122 .+-. 23
______________________________________
TABLE 7
______________________________________
Macrophage Activation by
Glycosidase-treated Cat DBPg
Ingestion Index
Dilution of Cat DBP
Glycosidase- untreated
Cat DBP treated with
Cat DBPg control .beta.-galactosidase
______________________________________
10.sup.-4 68 .+-. 26
320 .+-. 29
10.sup.-5 65 .+-. 23
275 .+-. 23
10.sup.-6 76 .+-. 20
108 .+-. 34
______________________________________
EXAMPLE 4
Purified DBP (1.0 .mu.g from each of the species identified in Table
8, below, was treated according to Example 3 with a mixture of
.beta.-galactosidase, sialidase and .alpha.-manosidase (0.5 U each)
in 1 ml of PBS-Mg containing 0.01M sodium phosphate, 0.9% NaCl and 1
mM MgSO.sub.4 for sixty minutes at 37.degree. C. The reaction
mixture containing each treated DBP was then diluted 10.sup.-4 in
0.1% supplemented EA medium and assayed for macrophage activation
activity according to the in vitro assay of Example 3B. The results
are set forth in Table 8. It may be observed that treatment with a
mixture containing all three enzymes resulted in conversion of DBP
to a potent macrophage activating factor, regardless of DBP
polymorphism.
TABLE 8
______________________________________
Ingestion Index
Treated with
Glycosidase- Untreated .beta.-galactosidase +
mannosidase control sialidase + .alpha.
______________________________________
Monkey (Macaca fucata)
72 .+-. 26
295 .+-. 38
Bovine (Bos taurus)
52 .+-. 19
320 .+-. 52
Sheep (Ovis aries)
48 .+-. 17
313 .+-. 48
Goat (Capra hircus)
56 .+-. 24
289 .+-. 32
Pig (Sus scrofa)
47 .+-. 12
332 .+-. 27
Horse (Equus caballus)
69 .+-. 23
266 .+-. 38
Cat (Felis catus)
58 .+-. 15
328 .+-. 43
Dog (Canis familigris)
60 .+-. 17
337 .+-. 18
Rat (Fisher) 65 .+-. 25
284 .+-. 37
Mouse (BALB/C)
71 .+-. 28
276 .+-. 34
______________________________________
EXAMPLE 6
A. Conversion of DBP to Macrophage Activating Factor with
Immobilized Enzyme
1. Preparation of Immobilized Enzymes
100 mg of CNBr-activated agarose (Sepharose 4B) was washed with 1mM
HCl and suspended in coupling buffer (300 .mu.l) containing
NaHCO.sub.3 buffer (0.1M, pH 8.3) and NaCl (0.5M). .beta.-Galactosidase,
.alpha.-Mannosidase or sialidase, or a combination of all three
enzymes (2 U each enzyme), were mixed in 600 .mu.l of the coupling
buffer and incubated at room temperature for 2 hours in an
end-over-end mixer. Remaining active groups in the agarose were
blocked by incubation with 0.2M glycine in coupling buffer for 2
hours at room temperature. The agarose-immobilized enzyme was washed
with coupling buffer to remove unabsorbed protein and glycine,
followed by washing with acetate buffer (0.1M, pH 4) containing NaCl
(0.5M), and additional coupling buffer. The agarose-immobilized
enzyme preparations were stored at 4.degree. C.
2 Conversion of DBP to Macrophage Activating Factor
DBP in 1 ml of PBS-Mg (pH 5.5) was combined with a mixture of the
above-prepared agarose-immobilized enzymes (2 units each enzyme) in
1 ml of PBS-Mg (pH 5.5). The reaction mixtures were incubated in 5
ml plastic tubes at 37.degree. C. in an end-over-end mixer for 30
minutes. The reaction mixtures were thereafter spun with a table-top
centrifuge at 2,000 rpm for 15 minutes. The supernatant of each
reaction mixture was collected, filtered through a sterilized
0.45.mu. pore size filter (type HA, Millipore Company, Bedford,
Mass.), and diluted.
B. In Vivo Assay of Macrophage Activating Factor
The enzymatically-modified DBP (100, 30, 10, 3 and 1 picogram
samples) were administered intramuscularly to BALB/c mice weighing
.about.20 grams. At 18 hours post-administration, peritoneal cells
were collected and placed on 12 mm coverglasses in the 16 mm wells
of tissue culture plates. The plates were incubated at 37.degree. C.
for 30 minutes to allow adherence of macrophages. The coverglasses
were washed in RPMI 1640 medium to dislodge non-adherent cells, and
then placed in new wells. Rabbit IgG-coated sheep erythrocytes as
prepared in Example 3B.2. were layered onto the coverglass, and a
phagocytosis assay was performed as in Example 3B.3. The results are
set forth in Table 9:
TABLE 9
______________________________________
In Vivo Assay of Macrophage Activation
by Glycosidase-treated Bovine DBPgs
Ingestion Index
Dosage of
Bovine DBP Dog DBP
enzymatically treated with treated with
modified .beta.-galacto- .beta.-galacto-
DBP (pico-
untreated
sidase and untreated
sidase and
gram/mouse)
control sialidase control
sialidase
______________________________________
100 63 .+-. 18
283 .+-. 42
55 .+-. 22
272 .+-. 29
30 56 .+-. 17
341 .+-. 38
43 .+-. 12
295 .+-. 35
10 52 .+-. 18
315 .+-. 44
63 .+-. 17
277 .+-. 41
3 51 .+-. 12
141 .+-. 27
51 .+-. 15
128 .+-. 27
1 65 .+-. 15
86 .+-. 12
60 .+-. 18
89 .+-. 26
______________________________________
| ( 1 of 1 ) |
| United States Patent | 5,837,252 |
| Sinnott , et al. | November 17, 1998 |
A nontoxic, therapeutic agent having pharmacological activity comprising concentrated extract of Larrea tridentata plant material and ascorbic acid, an ascorbic acid ester, an ascorbic acid salt, butylated hydroxyanisole, butylated hydroxytoluene, hydrogen sulfide, hypophosphorous acid, monothioglycerol, potassium bisulfite, propyl gallate, sodium bisulfite, sodium hydrosulfite, sodium thiosulfate, sulfur dioxide, sulfurous acid, a tocopherol, or vitamin E is made by a process in which the plant material is extracted using an organic solvent, preferably acetone, and is then saturated with one of the listed reducing agents acid to reduce the toxic NDGA quinone, which naturally occurs in the plant material, to NDGA itself. Additional amounts of ascorbic acid, an ascorbic acid ester, an ascorbic acid salt, butylated hydroxyanisole, butylated hydroxytoluene, hydrogen sulfide, hypophosphorous acid, monothioglycerol, potassium bisulfite, propyl gallate, sodium bisulfite, sodium hydrosulfite, sodium thiosulfate, sulfur dioxide, sulfurous acid, a tocopherol, or vitamn E may be added to the extract to inhibit the natural oxidation of the NDGA into the toxic NDGA quinone in vivo, or during processing or storage. The resulting extract is useful in the treatment of viral diseases caused by viruses from the Herpesviridae family or viruses which require the Sp1 class of proteins to initiate viral replications. The resulting compound can also be used as an anti-inflammatory when the inflaatory diseases are mediated by the effects of leukotrienes. The listed reducing agents can also be used to stabilize NDGA as a therapeutic agent or a food additive.
| Inventors: | Sinnott; Robert A. (Chandler, AZ); Clark; W. Dennis (Phoenix, AZ); DeBoer; Kenneth Frank (Belgrade, MT) |
| Assignee: | Larreacorp, Ltd. (Chandler, AZ) |
| Appl. No.: | 726686 |
| Filed: | October 7, 1996 |
| U.S. Class: | 424/195.1 |
| Intern'l Class: | A01N 065/00 |
| Field of Search: | 424/195.1 |
| 2373192 | Apr., 1945 | Lauer | 426/546. |
| 2382475 | Aug., 1945 | Gisvold | 568/729. |
| 2644822 | Jul., 1953 | Pearl | 549/435. |
| 4765927 | Aug., 1988 | Nomura et al. | 252/400. |
| 4774229 | Sep., 1988 | Jordan | 514/25. |
| 4880637 | Nov., 1989 | Jordan | 424/641. |
| 5276060 | Jan., 1994 | Neiss et al. | 514/731. |
Timmermann "Practical uses of Larrea,' in Creosote Bush: Biology and Chemistry of Larrea in the New World Deserts," Mabry et al. eds. (1977) (Dowden, Hutchison and Ross: Pennsylvania) pp. 252-276. Tierra, L., "Chapparal," The Herbs of Life: Health and Healing Using Western and Chinese Techniques, pp. 561, 180-181 (1992). Crellin, J.K. et al., "Chapparal," Herbal Medicines Past and Present II: A Reference Guide to Medicinal Plants, pp. 150-151 (1990). Sakakibara, et al., Flavonoid Methyl Esters on the External Leaf Surface of Larrea Tridentata and L. Divaricata, 1976, pp. 727-731. Obermeyer, et al., Chemical Studies of Phytoestrogens and Related Compounds in Dietary Supplements: Flax and Chaparral, 1995, pp. 6-10 vol. 208. Phytochemistry vol. 23(6) Donald MacRae, et al., Biological Activities of Lignans, 1984, pp. 1207-1220. Proc. Natl. Acad. Sci. USA vol. 92, Gnabre et al., Inhibition of human immunodeficiency virus type 1 transcription and replication by DNA sequence-selective plant lignans, 1995, pp. 11239-11243. Gnabre et al., Characterization of Anti-HIV Lignans from Larrea tridentata, 1995, pp. 12203-12210, Tetrahedron vol. 51(4). Gnabre et al., Isolation of anti-HIV-1 lignans from Larrea tridentata by counter-current chromatography, 1996, pp. 353-364 J. Chromatography vol. 719. Amer. Soc. Hematol. vol. 88(2) Nador et al., Primary Effusion Lymphoma: A Distinct Clinicopathologic Entity Associated With the Kaposi's Sarcoma--Associated Herpes Virus, 1996, pp. 645-656. The Philadelphia Inquirer Crenson, Kaposi's Sarcoma is tied to herpes, Jul. 31, 1996, p. A4. Altman, Aids Cancer Said to Have Viral Source: Breakthrough Seen in Kaposi's Sarcoma, Feb. 1, 1995, p. A22 The New York Times. Critchfield et al., Inhibition of HIV Activation in Latently Infected Cells by Flavonoid Compounds in AIDS Research and Human Retroviruses, 1996, pp. 39-46 vol. 12(1). Amer. College Phys. vol. 121 Henderson, The Role of Leukotrienes in Inflammation, 1994, pp. 684-697. Israel et al., Effect of Treatment With Zileuton, a 5-Lipoxygenase Inhibitor, in Patients With Asthma, 196, pp. 932-936 JAMA vol. 275(12). Gordon, The Microbicical Potential of Various Creosotebush (Larrea Tridentata) Extracts, 1987, pp. 83-85 vol. 21, J. Arizona--Nevada Acad. Sci. |
More recently, three scientific articles, John N. Gnabre et
al., Inhibition of human immunodeficiency virus type 1
transcription and replication by DNA sequence-selective plant
lignans, PROC. NATL. ACAD. SCI., USA, vol 92, pp.11239-11243
(November 1995); John Gnabre et al., Characterization of
Anti-HIV Lignans from Larrea tridentata, TETRAHEDRON, vol. 51,
pp. 12203-12210 (1995); and John Noel Gnabre et al., Isolation
of anti-HIV lignans from Larrea tridentata by counter-current
chromatography, JOURNAL OF CHROMATOGRAPHY A, vol. 719, pp.
353-364 (1996), the disclosure of all three articles being
incorporated herein by reference, demonstrate that at least two
lignans isolated from Larrea tridentata, NDGA and 3-O-methyl
nordihydroguaiaretic acid ("Mal. 4") inhibit
transcription and replication of human immunodeficiency virus
type 1 by a novel mechanism. This elucidated mechanism of
anti-HIV activity is thought to be due to the ability of the two
identified Larrea tridentata lignans, NDGA and Mal. 4, to
interfere with the binding of the Sp1 protein to Sp1 binding
sites in the HIV long terminal repeat (HIV-LTR). According to
this theory, by inhibiting Sp1 binding in the HIV-LTR, the
promoter activity of the HIV-LTR is eliminated so that HIV
transcription, HIV Tat-regulated transactivation and HIV
replication do not occur. It is further theorized by the authors
that viruses other than HIV, which require binding of Sp1
protein in promoter-containing Sp1 binding sites to initiate
viral replication, might also be inhibited by the anti-HIV
lignans isolated from Larrea tridentata and that this class of
lignans, in general, may possess a broader antiviral action of
important interest.
Also, in a recent article, Anneke K.Raney & Alan McLachlan,
Characterization of the Hepatitis B Virus Large Surface Antigen
Promoter Sp1 Binding Site, VIROLOGY, vol. 208, pp. 399-404
(1995), binding sites for the transcription factor Sp1 have been
identified in the DNA promoter regions of at least two important
viral genes of the Hepatitis B virus (HBV) which may be involved
in the coordinate regulation of HBV transcription by
transcription factor Sp1.
Kaposi's Sarcoma, a cancer that frequently occurs among AIDS
patients, has recently been implicated to be caused by a new
herpes virus, human herpes virus-8 (HHV-8). See Roland G. Nador
et al., Primary Effusion Lymphoma: A Distinct Clinicopathologic
Entity Associated With the Kaposi's Sarcoma--Associated Herpes
Virus, BLOOD, vol. 88, no. 2, pp. 645-656 (Jul. 15, 1996),
incorporated herein by reference. See also, Matt Crenson,
Kaposi's Sarcoma is tied to herpes, THE PHILADELPHIA INQUIRER,
p. A4 (Jul. 31, 1996), and Lawrence K. Altman, Aids Cancer Said
to Have Viral Source: Breakthrough Seen in Kaposi's Sarcoma, NEW
YORK TIMES .sctn. A, p. 22 (Feb. 1, 1995), incorporated herein
by reference.
Certain flavonoid compounds, especially members of the chemical
classes flavones and flavonols can inhibit HIV activation at
fairly low concentrations (See , J. William Critchfield et al,
Inhibition of HIV Activation in Latently Infected Cells by
Flavonoid Compounds in AIDS Research and Human Retroviruses,
AIDS RESEARCH AND HUMAN RETROVIRUSES, vol. 12, no. 1, pp. 39-46
(1996), incorporated herein by reference). As further cited in
Masayuki Sakakibara, et al., Flavonoid Methyl Ethers on the
External Leaf Surface of Larrea Tridentata and L. Divaricata in
PHYTOCHEMISTRY, vol. 15, pp. 727-731 (Pergamon Press 1976),
Larrea tridentata contains an abundance of these classes of
antiviral flavonoids, particularly methyl ethers of flavonols.
Like many physiologically active chemicals isolated from
plant sources, these antiviral lignans and flavonoid compounds
appear to work synergistically with other unresolved compounds
present in crude extracts of Larrea tridentata. The identity and
mode of action of these synergistic compounds is unknown but
they may facilitate absorption of the antiviral lignans or
otherwise enhance the specific physiological, antiviral effects.
NDGA is known to be a potent inhibitor of the enzyme
5-lipoxygenase. One of the enzymatic products of 5-lipoxygenase
is 5-hydroperoxyeicosatetraenoic acid (HPETE) which is the
precursor compound for the biosynthesis of very potent chemical
mediators of inflammation, known as leukotrienes. As detailed in
William R. Henderson, The Role of Leukotrienes in Inflammation,
ANN. INTER. MED., vol. 121, pp. 684-697 (1994), the disclosure
of which is incorporated herein by reference, 5-lipoxygenase is
limited to a specific number of myeloid cells including:
neutrophils, eosinophils, monocytes, macrophages, mast cells,
basophils and B-lymphocytes. Leukotrienes are chemicals which
induce prolonged muscle contraction, especially in the
bronchioles of the lungs, and also increase vascular
permeability and attract neutrophils and eosinophils to the site
of inflammation. The leukotrienes play a major role in the
inflammatory response to injury. Leukotrienes have also been
implicated in the pathogenesis of several inflamatory diseases
including: asthma, psoriasis, rheumatoid arthritis and
inflammatory bowel disease. The role of leukotrienes as
mediators of inflammatory diseases makes them attractive targets
for therapeutic drugs to treat these diseases.
Many inhibitors of leukotriene synthesis are being developed.
Recently, a 5-lipoxygenase inhibitor, Zileuton, was found to be
effective in the treatment of asthma during clinical tests
(Elliot Israel et al., Effect of Treatment With Zileuton, a
5-Lipoxygenase Inhibitor, in Patients With Asthma, JAMA, vol.
275, pp. 931-936 (Mar. 27, 1996), the disclosure of which is
incorporated herein by reference). The success of Zileuton
underscores the utility and need of therapeutic agents
containing 5-lipoxygenase inhibitors in the treatment of
inflammatory disease processes, including asthma
Throughout this specification and claims, viral diseases are
intended to include all diseases, attributed to a pathological
virus of humans or animals, in which the causative viral agent
which requires the Sp1 class of proteins to initiate viral
replication, including certain viral agents of venereal diseases
such as the Herpes viruses (the Herpesviridae), HSV-1 and HSV-2,
viral hepatitis (the Hepadnaviridae) such as hepatitis B, and
members of the retrovirus family (the retroviridae) including
Varicella-Zoster viruses, cytomegalovirus (CMV), the human T-lymphotrophic
viruses 1 and 2 (HTLV-1 and (HTLV-2) the human immunodeficiency
viruses 1 and 2 (HIV-1 and HIV-2) and the cancer Kaposi's
Sarcoma. Inflammatory diseases, throughout the specification and
claims, are intended to include all diseases in which
leukotrienes are known to play a major role or have been
implicated including: asthma, allergic rhinitis, psoriasis,
rheumatoid arthritis, inflammatory bowel disease, inflammatory
pain, cystic fibrosis, adult respiratory distress syndrome,
glomerulonephritis, inflammation of the skin, and virally
induced inflammation (caused by CMV and other members of the
Herpesviridae) leading to atherosclerosis/ arteriosclerosis and
subsequent coronary artery disease.
In light of the foregoing background, there exists the need for
a commercial method of producing a Larrea tridentata extract
which contains a high concentration of both the identified
antiviral lignans (NDGA and Mal. 4), flavonoids, and a wide
variety of other associated organic compounds from the leaf
resin, which may contribute synergistic antiviral and
lipoxygenase inhibitory activity.
Additionally, for the purpose of toxicological safety, it is
of critical importance that the Larrea tridentata extract, to be
used for medical applications, be processed to reduce the
concentration of the toxic compound, NDGA quinone, which is
reported to occur naturally in Larrea tridentata plant tissues.
There is also a need to inhibit the natural production of toxic
oxidation products, such as NDGA quinone, in the Larrea
tridentata extract during processing and storage of the extract
and formulated products and to facilitate the processing of the
concentrated extract as either a liquid, slurry, or solid.
Lastly, there is a need for products comprising synthesized NDGA
which is stabilized against oxidation into NDGA quinone during
processing and storage.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to meet
the above described needs and others. It is an object of the
present invention to provide a nontoxic extract of Larrea
tridentata having a high concentration of NDGA. It is a further
object of the present invention to provide a nontoxic extract of
Larrea tridentata containing Mal.4 and other compounds having
known or expected, and perhaps synergistic, therapeutic effects.
It is a further object of the present invention to provide such
an extract which contains little or no NDGA quinone and which
inhibits the production of NDGA quinone in vivo and during
processing and storage. It is a further object of the present
invention to provide a method of making the above-described
extract, and formulations thereof. It is a further object of the
present invention to describe some of the potential therapeutic
uses of the above-described extract, and formulations thereof.
It is a further object of the present invention to provide NDGA
products for use as food additives or therapeutic agents in
which the NDGA is prevented during storage and processing from
oxidizing into NDGA quinone.
Additional objects, advantages and novel features of the
invention will be set forth in the description which follows or
may be learned by those skilled in the art through reading these
materials or practicing the invention. The objects and
advantages of the invention may be achieved through the means
recited in the attached claims.
To achieve the stated and other objects of the present
invention, as embodied and described below, the invention may
comprise:
A method of preparing a nontoxic extract of Larrea tridentata
plant material comprising the steps of:
extracting endogenous antiviral and anti-inflammatory lignans
and flavanoids and other synergistic compounds from Larrea
tridentata plant material with a polar solvent, preferably
acetone, to produce an extract by recirculating the solvent over
the plant material a plurality of times;
filtering the extract;
adding an emulsifying and stabilizing agent, preferably
polysorbate 80, to the filtered extract;
reducing the NDGA quinone in the extract with ascorbic acid,
ascorbic acid esters (i.e. ascorbyl palmitate), ascorbic acid
salts (i.e. sodium ascorbate), butylated hydroxyanisole (BHA),
butylated hydroxytoluene (BHT), hydrogen sulfide,
hypophosphorous acid (phosphinic acid), monothioglycerol (3
mercapto-1,2-propanediol), potassium bisulfite potassium
metabisulfite, potassium pyrosulfite), propyl gallate, sodium
bisulfite (sodium metabisulfite, sodium pyrosulfite), sodium
hydrosulfite (sodium dithionite), sodium thiosulfate (sodium
hyposulfite), sulfur dioxide, sulfurous acid, a tocopherol, or
vitamin E (DL-alpha-tocopherol), preferably ascorbic acid, by
passing the extract through a bed of ascorbic acid powder;
boiling the organic solvent out of the compound; and
adding additional amounts of one of the above listed reducing
agents to the compound subsequent to the step of reducing.
The present invention may also comprise:
A nontoxic, therapeutic agent comprising:
an extract of Larrea tridentata plant material comprising NDGA
and Mal.4; and
ascorbic acid, ascorbic acid esters (i.e. ascorbyl palmitate),
ascorbic acid salts (i.e. sodium ascorbate), butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT), hydrogen
sulfide, hypophosphorous acid (phosphinic acid),
monothioglycerol (3 mercapto-1,2-propanediol), potassium
bisulfite (potassium metabisulfite, potassium pyrosulfite),
propyl gallate, sodium bisulfite (sodium metabisulfite, sodium
pyrosulfite), sodium hydrosulfite (sodium dithionite), sodium
thiosulfate (sodium hyposulfite), sulfur dioxide, sulfurous
acid, a tocopherol, or vitamin E (DL-alpha-tocopherol).
The present invention may also comprise:
formulations of the described therapeutic agent in the
embodiment of a lotion, liquid, powder or pill.
The present invention may also comprise a food additive or
therapeutic agent comprising:
NDGA; and
ascorbic acid, ascorbic acid esters (i.e. ascorbyl palmitate),
ascorbic acid salts (i.e. sodium ascorbate), butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT), hydrogen
sulfide, hypophosphorous acid (phosphinic acid),
monothioglycerol (3 mercapto-1,2-propanediol), potassium
bisulfite (potassium metabisulfite, potassium pyrosulfite),
propyl gallate, sodium bisulfite (sodium metabisulfite, sodium
pyrosulfite), sodium hydrosulfite (sodium dithionite), sodium
thiosulfate (sodium hyposulfite), sulfur dioxide, sulfurous
acid, a tocopherol, or vitamin E (DL-alpha-tocopherol)
The present invention may also comprise:
A method of treating:
Viral diseases caused by viruses from the retrovirus family of
viruses, the Hepatitis B virus, the Herpesviridae family of
viruses, and viruses which require Sp1 class proteins to
initiate viral replication,
inflammatory diseases which are mediated by the effects of
leukotrienes, and virally induced inflammation leading to
atherosclerosis, hypertension, atherosclerosis,
arteriosclerosis, and subsequent coronary artery disease
using a nontoxic, therapeutic agent comprising:
an extract of Larrea tridentata plant material comprising NDGA
and Mal. 4; and
ascorbic acid, ascorbic acid esters (i.e. ascorbyl palmitate),
ascorbic acid salts (i.e. sodium ascorbate), butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT), hydrogen
sulfide, hypophosphorous acid (phosphinic acid),
monothioglycerol (3-mercapto-1,2-propanediol), potassium
bisulfite (potassium metabisulfite, potassium pyrosulfite),
propyl gallate, sodium bisulfite (sodium metabisulfite, sodium
pyrosulfite), sodium hydrosulfite (sodium dithionite), sodium
thiosulfate (sodium hyposulfite), sulfur dioxide, sulfurous
acid, a tocopherol, or vitamin E (DL-alpha-tocopherol).
BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings illustrate the present invention and
are a part of the specification. Together will the following
description, the drawings demonstrate and explain the principles
of the present invention. In the drawings:
FIG. 1 is a high performance liquid chromatography
("HPLC") tracing of NDGA, as a purified compound
(reported by the manufacturer to have a minimum purity of 90%).
Slight amounts of unidentified impurities, probably methylated
NDGA compounds, are indicated. However, no NDGA quinone is
detectable in the sample. The tracing was taken at 280 nm
absorbance.
FIG. 2 is a HPLC tracing of NDGA as a purified compounds
described in reference to FIG. 1 which has been treated with the
strong oxidizing agents sulfuric acid and potassium dichromate.
NDGA quinone, the oxidation product of NDGA, is identified in
the chromatogram as the large peak at 16.5 minutes. The tracing
was taken at 280 nm absorbance.
FIG. 3 is an HPLC tracing of a purified NDGA sample as described
in reference to FIG. 1 and treated with oxidizing agents as
described in reference to FIG. 2, and then treated according to
the principles of the present invention, with the chemical
reducing agent, ascorbic acid. NDGA quinone is not detectable at
16.5 minutes after treatment was completed. The tracing was
taken at 280 nm absorbance.
FIG. 4 is an HPLC tracing of a concentrated Larrea tridentata
extract, which has been treated with the strong oxidizing agents
sulfuric acids and potassium dichromate. A peak corresponding
with NDGA quinone is identified in the chromatogram as the small
peak occurring at 30 minutes. The tracing was taken at 375 nm
absorbance.
FIG. 5 is an HPLC tracing of a concentrated Larrea tridentata
extract, produced according to the methods of the present
invention. No NDGA quinone peak is observed at 30 minutes. The
tracing was taken at 375 nm absorbance.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The present invention includes at least two products.
(1) a nontoxic extract of Larrea tridentata plant material
having a high concentration of NDGA and little or no NDGA
quinone which can be used as a therapeutic agent; and
(2) NDGA, for use as a food additive or therapeutic agent, which
does not oxidize into NDGA quinone during storage or processing.
An important principle of the invention is that ascorbic acid,
ascorbic acid esters (i.e. ascorbyl palmitate), ascorbic acid
salts (i.e. sodium ascorbate), butylated hydroxyanisole (BHA),
butylated hydroxytoluene (BHT), hydrogen sulfide,
hypophosphorous acid (phosphinic acid), monothioglycerol (3
mercapto-1,2-propanediol), potassium bisulfite (potassium
metabisulfite, potassium pyrosulfite), propyl gallate, sodium
bisulfite (sodium metabisulfite, sodium pyrosulfite), sodium
hydrosulfite (sodium dithionite), sodium thiosulfate (sodium
hyposulfite), sulfur dioxide, sulfurous acid, a tocopherol, or
vitamin E (DL-alpha-tocopherol), when combined with NDGA, reduce
any NDGA quinone present into NDGA, and prevent the NDGA from
oxidizing and producing more NDGA quinone. These agents are not
necessarily considered to be equivalents to each other, some
having advantages not possessed by all the others. It is only
asserted that each of these listed agents may be used according
to the principles of the invention as described in this
specification to substantially accomplish the objects of the
invention. The preferred embodiment of the invention will be
described below.
According to the principles of the present invention, a nontoxic
extract of Larrea tridentata having a high concentration of NDGA
and very little or no NDGA quinone can be prepared by saturating
the extract with one or more of the above listed agents.
Additional amounts of one of the listed agents may be added to
the extract or products formulated therefrom to inhibit the
natural oxidation of component NDGA into NDGA quinone during
processing and storage.
According to the preferred embodiment of the present invention,
a nontoxic extract is prepared by the following method. Larrea
tridentata plant material consisting mostly of leaves and stems,
but also possibly containing a small amount of flowers and
fruits, i.e. whole plant material, is air dried. The dried plant
material is extracted using a suitable solvent. In the preferred
embodiment, the solvent is an organic solvent, preferably
acetone.
The organic solvent is recirculated three times over the correct
ratio of plant material to completely dissolve the organic
compounds on the surface of the plant material and to produce a
crude Larrea tridentata extract. The resulting crude extract is
filtered through cellulosic media (i.e. qualitative grade filter
paper) to remove dirt and particulates. A chemical emulsifying
and stabilizing agent, Food Chemicals Codex (F.C.C.) grade
polysorbate 80, is added to the clarified extract at a
concentration of 0.01% by volume.
The clarified extract is then passed through a bed of ascorbic
acid powder (5 grams of F.C.C. grade ascorbic acid powder for
each liter of extract passed through) in a manner which
facilitates contact of the extract with the ascorbic acid powder
and results in saturation of the extract with ascorbic acid.
Saturating the extract with the chemical reducing agent,
ascorbic acid results in conditions which favor the chemical
reduction of the toxic, oxidative metabolites of NDGA, which are
present in Larrea tridentata plant tissues and the resulting
extracts. By chemically reacting with the ascorbic acid, the
toxic NDGA quinones are reduced to NDGA hydroquinone which is
NDGA itself.
To concentrate the Larrea tridentata organic extract which is
saturated with ascorbic acid, the extract is transferred to a
water jacketed, stainless steel tank, which is heated to
approximately 100 degrees C. by circulation of a suitable
solvent (i.e. water). While the extract is heated in the tank,
the organic solvent is boiled out of the extract and may be
recovered by condensation for reuse. The extract is heated in
the tank to approximately 10 degrees above the boiling point of
the organic extraction solvent. This rise in temperature of the
extract indicates that the concentrated Larrea tridentata
extract is substantially free of the extraction solvent The
concentrated extract, approximately 1/10 the volume of the
original Larrea tridentata extract, is then removed from the
tank and packaged in tightly sealed plastic drums for storage
and shipping.
Another aspect of the present invention includes mixing the
concentrated Larrea tridentata extract with effective,
additional amounts of ascorbic acid, ascorbic acid esters (i.e.
ascorbyl palmitate), ascorbic acid salts (i.e. sodium
ascorbate), butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), hydrogen sulfide, hypophosphorous acid (phosphinic
acid), monothioglycerol (3 -mercapto-1,2-propanediol), potassium
bisulfite (potassium metabisulfite, potassium pyrosulfite),
propyl gallate, sodium bisulfite (sodium metabisulfite, sodium
pyrosulfite), sodium hydrosulfite (sodium dithionite), sodium
thiosulfate (sodium hyposulfite), sulfur dioxide, sulfurous
acid, a tocopherol, or vitamin E (DL-alpha-tocopherol).
Formulation of the extract with excess amounts of one or more of
the listed reducing agents prevents subsequent formation of NDGA
quinone during subsequent processing, storage, and formulating
of the extract, and functions as a synergistic antioxidant in
vivo with NDGA and other endogenous antiviral compounds.
Our invention is based in part on our discovery that a Larrea
tridentata extract, which is produced according to the methods
of our invention, contains no detectable amount of NDGA quinone
when analyzed by high performance liquid chromatography. Because
of the foregoing discussion linking NDGA quinone with the toxic
effects associated with the consumption of Larrea tridentata
products, the production of Larrea tridentata extract, with a
very low concentration of NDGA quinone, for use in medical
formulations is very important.
Further, we have found through experiments performed by the
inventors, that formulations based on the Larrea tridentata
extract and ascorbic acid compound, as described in this
invention, have pronounced antiviral activity against Herpes
simplex virus types 1 and 2, and against Kaposi's Sarcoma in
human patients. We have also found through experimentation that
the described formulations have pronounced antiviral activity
against Herpes simplex virus type 1 (HSV-1) in both animal cell
culture models and human volunteers as well as anti-inflammatory
action in human volunteers.
The present invention therefore provides methods for the
medically useful and effective extract of Larrea tridentata and
formulations for production of pharmaceutical agents which have
immediate and commercially important utility in the medical
treatment of viral and inflammatory diseases.
Experimental Results
The following are provided by way of illustrating examples of
extraction and processing formulations of the Larrea tridentata
extract, and are provided by way of illustration only and are
not intended to limit the invention in any way.
EXAMPLE 1
Plant material (consisting of mostly leaves but with some small
branches and a small amount of fruits and flowers) was harvested
from Larrea tridentata shrubs growing in the Arizona desert,
southwest of Gila Bend, Arizona. The plant material was air
dried in the shade at ambient temperature and humidity for one
week before processing. 100 kg of plant material was extracted
with 100 liters of F.C.C. grade acetone by recirculating the
acetone over the plant material three times. The resulting
Larrea tridentata extract was filtered through Whatman #1 filter
paper.
5 mils of F.C.C. grade polysorbate 80 was added to 50 liters of
the extract and the extract was slowly passed through a glass
column packed with 250 g of powdered ascorbic acid. The extract,
saturated with ascorbic acid, was concentrated by boiling off
the acetone solvent in a water-jacketed stainless steel tank
which was heated to approximately 100 degrees Celsius by
circulating hot water. The resulting concentrated extract, in
the form of a viscous liquid, was collected in plastic drums and
used to prepare medical formulations.
EXAMPLE 2
Several formulations of concentrated Larrea extract suitable for
encapsulation were prepared by thoroughly mixing the
concentrated extract, as produced in example 1, with dry
excipient materials including starch, sucrose, fructose, and
ascorbic acid powder. In some cases, other ingredients including
antiviral and anti-inflammatory agents or herbal extracts i.e.
Echinacea, Podophyllin, etc. were also combined in formulations.
In all cases, the mixtures consisted of one part concentrated
Larrea tridentata extract with 5 to 10 parts dry excipient
material. These formulations were used to treat HIV
opportunistic infections, and Herpes virus infections described
in this specification. The treatment consisted of administering
several capsules containing 50 to 100 mg of Larrea tridentata
extract per capsule which were ingested daily.
EXAMPLE 3
Several formulations of concentrated Larrea tridentata extract
in a lotion base were prepared by adding the concentrated
extract, as produced in example 1, into various lotion
formulations. The concentration of extract added to the lotion
base can range from 0.1 to 5% volume/volume. In initial trial
with human volunteers, a 3% lotion prepared according to the
principles of the present invention has been used successfully
in the medical treatment of athletes foot, viral lesions caused
by herpes simplex virus (HSV-1, HSV-2), Kaposi's Sarcoma, and
inflammation of the skin induced by contact allergens,
ultraviolet light and thermal exposure. The treatment consisted
of applying the lotion frequently and liberally to the affected
areas.
EXAMPLE 4
Results obtained by the inventors show that the concentrated
Larrea extract, produced by the methods of the present
invention, when used at a concentration greater than 20
micrograms per milliliter, is nearly 100 % effective in
protecting African Green Monkey kidney cells from destruction by
the Herpes simplex-1 virus (HSV-1).
It is apparent from the evidence presented above that the
methods and compositions of the present invention meet
long-standing needs in the medical treatment of viral and
inflammatory diseases.
Many alternatives to the most preferred embodiment, described
above, are also part of the present invention. For example, when
the NDGA quinone is reduced using ascorbic acid, ascorbic acid
esters (i.e. ascorbyl palmitate), ascorbic acid salts (i.e.
sodium ascorbate), butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), hydrogen sulfide, hypophosphorous acid (phosphinic
acid), monothioglycerol (3 -mercapto-1,2-propanediol), potassium
bisulfite (potassium metabisulfite, potassium pyrosulfite),
propyl gallate, sodium bisulfite (sodium metabisulfite, sodium
pyrosulfite), sodium hydrosulfite (sodium dithionite), sodium
thiosulfate (sodium hyposulfite), sulfur dioxide, sulfurous
acid, a tocopherol, or vitamin E (DL-alpha-tocopherol) in a
Larrea tridentata extract, it is not essential to add further
amounts of one of these listed reducing agents as described.
While doing so provides a more stable nontoxic product, if
additional amounts of one or more of these listed reducing
agents are not added, the resulting extract will still have use
as a nontoxic therapeutic agent.
Moreover, it is not essential to use any of the listed reducing
agents to reduce the NDGA quinone present initially in the
extract. If another, perhaps even toxic, reducing agent is used
to reduce the NDGA quinone naturally occurring in the Larrea
tridentata extract, ascorbic acid, ascorbic acid esters (i.e.
ascorbyl palmitate), ascorbic acid salts (ie. sodium ascorbate),
butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),
hydrogen sulfide, hypophosphorous acid (phosphinic acid),
monothioglycerol (3 -mercapto-1, 2-propanediol), potassium
bisulfite (potassium metabisulfite, potassium pyrosulfite),
propyl gallate, sodium bisulfite (sodium metabisulfite, sodium
pyrosulfite), sodium hydrosulfite (sodium dithionite), sodium
thiosulfate (sodium hyposulfite), sulfur dioxide, sulfurous
acid, a tocopherol, or vitamin E (DL-alpha-tocopherol) can then
be added to the resulting product to produce a nontoxic extract
which will not develop quantities of NDGA quinone through
oxidation during processing and storage. In such a process, an
additional step to remove the reducing agent would likely be
necessary.
Finally, as noted in the prior art NDGA has many known uses as
an antioxidant food additive and as a therapeutic agent. NDGA
for these purposes can be produced by extraction from natural
sources, such as the Larrea tridentata, or can be synthesized.
However the NDGA is produced, during processing and storage it
oxidizes to produce NDGA quinone and is therefore toxic when
ingested. Accordingly, it is within the scope of the invention
to combine NDGA, whether extracted or synthesized, with ascorbic
acid, ascorbic acid esters (i.e. ascorbyl palmitate), ascorbic
acid salts (ie. sodium ascorbate), butylated hydroxyanisole (BHA),
butylated hydroxytoluene (BHT), hydrogen sulfide,
hypophosphorous acid (phosphinic acid), monothioglycerol (3
-mercapto-1,2-propanediol), potassium bisulfite (potassium
metabisulfite, potassium pyrosulfite), propyl gallate, sodium
bisulfite (sodium metabisulfite, sodium pyrosulfite), sodium
hydrosulfite (sodium dithionite), sodium thiosulfate (sodium
hyposulfite), sulfur dioxide, sulfurous acid, a tocopherol, or
vitamin E (DL-alpha-tocopherol) to produce a stable, nontoxic
NDGA product that can be used as a food additive or therapeutic
agent.
The preceding description has been presented only to illustrate
and describe the invention. It is not intended to be exhaustive
or to limit the invention to any precise form disclosed. Many
modifications and variations are possible in light of the above
teaching.
The preferred embodiment was chosen and described in order to
best explain the principles of the invention and its practical
application. The preceding description is intended to enable
others skilled in the art to best utilize the invention in
various embodiments and with various modifications as are suited
to the particular use contemplated. It is intended that the
scope of the invention be defined by the following claims.
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