Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
W093/2~239 PCT/US92/05091
21~5295
ARABINOGAL~CTAN DERIVATIVES AND USES THEREOF
S Technical Field
This invention relates to the synthesis and methods of
use of therapeutic agents targeted to cells, especially
hepatocytes.
Backaround Art
The safety and efficacy of a therapeutic agent is a
function of (i) its intrinsic biological acti~ity and ~ii)
the biodistribution achieved after its administration. Many
potentially useful therapeutic agents possess a biochemical
activity ameliorating a par~icular pathological condition,
lS but the presence of the agent in normal, nonpathological
tissue results in deleterious effects that prevent the use
of the agent. Damage to a normally functioning kidney, bone
marrow, liver tissue or other organ may limit`the u e of
therapeutic agents with established antiviral activity, or
20 agents with established anti-cancer activity. There is a
need for new compounds to target therapeutic agents to the
specific cells that are the source of some pathological
condition, and to reduce the concentration attained in
unaffected, normal tissues. Targeting is the modification
25 of a therapeutic agent so that after injection or oral
administration the uptake by a specific population of cells
is increased re1ative to uptake of the unmodified agent. By
targeting compounds with established and beneficial
biological activity to specific tissues, compounds whose use
is currently limited by side effects might become safe and
, efficacious drugs. A therapeutic agent is a compound
administered with the intent of changing in a beneficial
manner some physiological function. Therapeutic agents
include radioprotective agents, chemoprotective agents,
35 antiviral agents, antibodies, enzymes, and peptides.
One method of targeting therapeutic agents to specific
cells involves attaching them to carrier molecules
recognized by receptors performing receptor mediated
endocytosis. Of particular interest is targeting via the
W093~2~239 2 1 3 5 ~ 3 ~ PCT/US92/0509l
asialoglycoprotein receptor of hepatocytes. This receptor
is present in high levels on normal hepatocytes but in lower
levels or not at all on transformed hepatocytes (hepatoma
cells). Diagnostic and therapeutic agents have been
5 attached to asialoglycoprotein carriers and neoglycoprotein
carriers recognized by the asialoglycoprotein receptor and
- targeted to the cells, see Table II of Meijer and van der
Sullies, Pharm. Res. (1989) 6:105-118 and Ranade, J. Clin.
Pharmacol. (1989) 29:685-694. Molecules recognizing the
10 asialoglycoprotein receptor are most often either
asialoglycoproteins or neoglycoproteins.
Asialoglycoproteins are formed by removing the sialic acid
of glycoproteins and exposing galactose residues.
Neoglycoproteins are formed by attaching multiple galactose
15 residues to non-glycoproteins such as human albumin.
When attaching diagnostic and therapeutic agents to a
receptor-recognizing carrier molecule, targeting can be
achieved only if the affinity of the carrier for the
receptor is maintained. The differential reactivity of the
20 protein amine and carbohydrate hydroxyl groups of
glycoprotein carriers, e.g. asialofetuin, i5 commonly used
to achieve this goal. The highly reactive amine groups of
~, .
protein lysine residues are selectively modified, while the
hydroxyl groups of carbohydrate are left intact and continue
25 to recognize the receptor. Examples of this strategy are
given in Van der Sluijs et al. (above~ and in "Liver
Diseases, Targeted Diagnosis and Therapy Using Specific
Receptors and Ligands" (1991) Ed. G.Y. Wu and C.H. Wu,
Marcel Dekker Inc. pp. 235-264. In contrast, a
30 polysaccharide such as arabinogalactan offers no polypeptide
~ amino groups distal from the receptor binding site that can
; be modified for the purposes of retaining the
asialoglucoprotein receptor binding activity. In spite of
the obvious strategy for modification of glycoproteins with
35 retention of receptor binding activity, their use for
targeted, parenteral pharmaceuticals is subject to several
problems.
.
W093/25239 2 1 3 5 2 9 S PCT/US92/05091
-- 3
(i) Glycoproteins are prepared from animal cells and
insuring noncontamination with human infective viral
pathogens is a major issue.
(ii) Glycoproteins will not generally tolerate organic
5 solvents during conjugate synthesis, because such solvents
frequently lead to a loss of biological activity and
denaturation.
(iii) Glycoproteins can be toxic and/or antigenic.
(iv) Glycoproteins in their native form, e.g. fetuin,
10 do not afford galactose resides and must be desialylated to
produce a carrier which interacts with the receptor.
Arabinogalactans are a class of polysaccharides
obtained from the cell walls of many species of trees and
other plants. A common, commercially available source of
15 arabinogalactan is the American Western larch (Larix
occidentalis). Arabinogalactan from this source is used as
a binder, emulsifier or stabilizer in foods. It consists ~f
a largely 1-3 linked D-galactose bac~bone with 1-6 linked
branch chains of L-arabinoses and D-galactoses at
20 practically every residue on the backbone. In larch
arabinogalactans the ratio of galactose to arabinose is
between 5 to 1 and 10 to l, while arabinogalactans from
plant sources in general range from about 1 to 4 to about 10
to l [Clarke, A.E., Anderson, R.L., Stone, B.A.;
25 Phytochemistry (1979) 18: 521-40~. Like many
polysaccharides, arabinogalactans have different molecular
weights with values of about 1-2 million to a~out 10,000
daltons ~Blake, J.D., Clarke, M.L., Jansson, P.E., Carbohydr
Res tl9833 115: 265-272] having been reported. It has been
30 shown that L-arabinose and D-galactose interact with the
asialoglycoprotein receptor while common monosaccharides
like glucose or mannose do not [Lee, Haekyung, Kelm, Sorge,
Teruo, Yoshino, Schauer, Roland Biol. Chem., Hoppe-Seyler
(1988) 369, 705-714].
Some derivatives of arabinogalactan have been
previously prepared. Graft copolymers have been used in
paper manufacturing [SUl285094] and in soil treatments
WO 93f25239 2 1 3 5 ~ .S PCI/US92/0509~
-- 4
[JP1051198]. Arabinogalactan sulfate has been used to form
salts with drugs ~o influence drug absorption and prolong
drug action [US4609640]. Acidic forms of arabinogalactan
occur naturally having a composition which includes uranic
5 acid ~Clarke, A.E., Anderson, R.L., Stone, B.A.,
Phytochemistry (1979) 18, 521-40], and have also been
prepared from arabinogalactan [JP60219202]. Derivatives of
arabinogalactan with substituent alkyl, allyl cyano, halo or
amino groups, and conjugates with organic acids and enzyme
10 protein have been disclosed, wherein the carbohydrate is
used as a carrler, adsorbent or resin ~JP60219201]. In some
cases arabinogalactan has been highly derivatized in a
manner likely to destroy its interaction with the
asialoglycoprotein receptor. For example, in some cases as
15 many as 50% of the hydroxyl groups of arabinogalactan have
been modified ~JP60219201], but the affinity, or lack
thereof, of arabinogalactan derivatives for the
asialoglycoprotein receptor has not been studied.
Summary of Invention
The present invention provides for derivatives of
arabinogalactan which can be used to target therapeutic
- agents to the cells possessing the asialoglycoprotein
receptor.
~- The use of the polysaccharide arabinogalactan to target
25 therapeutic agents to cells via the asialoglycoprotein
receptor, a feature of the current invention, overcomes
problems encountered when glycoproteins are used for this
,
purpose.
(i) A polysaccharide like arabinogalactan originating
30 from a plant source is unlikely to be contaminated with
human viral pathogens.
(ii) Since arabinogalactan is a polysaccharide, it
will tolerate exposure to organic solvents, which normally
denature proteins during conjugate synthesis. Composed
35 exclusively of sugars, the polysaccharide presents a narrow
; spectrum of reactive sites, an advantage compared to
proteins where the variety of reactive sites can lead to
W093/25239 2 1 3 5 2 ~ 5 P~T/US92~05091 '
-- 5
unwanted synthetic byproducts. This advantage is evident in
the examples below.
(iii) Arabinogalactan has low toxicity and
antigenicity.
(iv) Arabinogalactan in its natural form reacts with
the asialoglycoprotein receptor. This helps reduce
manufacturing cost because the deasialylation reaction
normally used to expose the penultimate galactose of
glycoproteins is avoided.
We have discovered that arabinogalactan can be modified
in a number of ways to produce molecules which preserve the
useful affinity for the asialoglycoprotein receptor. This
is surprising since arabinogalactan does not afford protein
or amino groups for selective modifications distal from the
15 receptor binding site. The ability to modify
arabinogalactan while retaining its biological activity
permits its use as a carrier for a wide variety of
therapeutic agents with various targeting strategies.
In some instances targeting may be employed to deliver
20 a therapeutic agent to normal rather than pathological
tissue. This strategy is employed when it is desirable to
protect normal tissues from other generally toxic agents; in
some cases agents of known but controlled toxicity are
employed in therapy. The targeting of protective agents
25 used in conjunction with normally toxic radiation, as in
radiation therapy, is an embodiment of the current invention
and example of this type of targeting. The targeting of
protective agents used with chemotherapeutic agents used in
cancer treatment is another embodiment of the current
invention. The use of the term "therapeutic agent" in this
description and the accompanying claims, includes agents
which are protective from toxic chemicals or radiation.
The arabinogalactan derivatives of the invention must
interact strongly with the asialoglycoprotein receptor, so
35 they can be used to target therapeutic agents to cells via
that receptor. An assay to determine the strength of the
interaction of arabinogalactan derivatives of the invention
i~
W093/2~239 ~ PCT/US92/0509l
2~ 3~29`-~ 6 -
with the receptor is presented.
In one embodiment the antiviral therapeutic agent
adenosine arabinoside mono-5'-phosphate (ARA-AMP) is coupled
to arabinogalactan. In addition, ARA-A or acyclovir, both
5 antiviral therapeutic agents, may also separately be coupled
to arabino-galactans. In another embodiment the
radioprotective agent S-2-(3 aminopropylamino) ethyl-
thiophosphoric acid (known as WR2721) is attached to
arabinogalactan. The invention provides methods and
10 compositions which enable the attachment of a variety of
therapeutic agents to arabinogalactan and the delivery of
those agents into the cytoplasm of cells via endocytotic
activity of the asialoglycoprotein receptor.
Detailed Descri~tion of Specific Embodiments
The arabinogalactan used here in a preferred embodiment
is highly purified and substantially free of endotoxins, and
is derived from the Western Larch and has a single peak by
size exclusion chromatography of about 20,000 daltons.
Arabinogalactan can be used in its native, 20,000 dalton
form; alternatively polymers of arabinogalactan (molecular
weight greater than the 20,000 dalton form), or degradative
products (molecular weight below the 20,000 dalton form) can
be used~ Purified arabinogalactan has a single peak of
20,000 daltons by gel filtration, and a ratio of galactose
25 to arabinose of 5 to 1 as determined by the alditol acetate
method. It binds the asialoglycoprotein receptor on
h~patocytes tJosephson Groman et al. Mag. Res. Imag. (1990)
8: 637-646]. It has been shown that L-arabinose, and D-
galactose interact with the asialoglycoprotein receptor
30 while, for example, common monosaccharides like glucose or
mannose do not [Lee, Haekyung, Kelm, et al., Biol. Chem.,
~oppe-Seyler (1988) 369: 705-714]. It has also been shown
that an underivatized 4-hydroxy group on galactose and the
clustering of suitable sugars, as is displayed by highly
35 branched polysaccharides like arabinogalactan, are important
factors in binding the asialoglycoprotein receptor. Given
these requirements, and based on the above composition and
W093/25239 2 1 ~ ~ 2 ~ 5 PCT/US92/oSOgl
-- 7
structure, arabinogalactan is distinguishable from other
polysaccharides including dextrans, starches, celluloses,
inulins, 1-4 linked galactan and gum arabi Though
chemically distinguishable from arabinogalactan, gum arabic
5 is another polysaccharide which like arabinogalactan
interacts with the asialoglycoprotein receptor.
The present invention provides for conjugates of
arabinogalactan with the therapeutic agents such as ARA-AMP
or WR2721. The present invention also provides derivatives
10 of arabinogalactan which interact with the
asialoglycoprotein receptor. When an ara~inogalactan
derivative is recognized by the asialoglycoprotein receptor,
a therapeutic agent zan be targeted into the cells
possessing that receptor, chiefly the hepatocytes.
15 Asialoglycoprotein receptors are dramatically reduced in
primary hepatocellular cancers, and totally absent in
secondary cancers to the liver, but are found in high
concentration on normal hepatocytes [Josephson, Groman et
al., Mag. Res. Imag. (1990) 8:637-646]. Hepatocytes are the
20 predominant cell possessing this receptor, and endocytose a
large proportion of injected radiolabelled
asialoglycoproteins [Hub~ard, Wilson et. al., J. Cell Biol.
(1979) 83:47-64]. However, asialoglycoprotein receptors
have been detected on Kupffer cells ~Lee, Haekyung, et al.
25 Biol. Chem. Hoppe-Seyler (1988) 369: 705-714~, bone marrow
cells ~Samoloski and Daynes, Proc. Nat. Acad. Sci. (1985)
82:2508-2512] and rat testis ~Abullah and Kierszenbaum, J.
Cell Biol. (1989) 108: 367-375]. Useful amounts of a
therapeutic agent may be targeted to any asialoglycoprotein
30 receptor positive cell. Similarly any receptor positive
cell, including stem cells, may be protected with a receptor
targeted radioprotective agent based on arabinogalactan. ?
ARA-AMP is an antiviral therapeutic agent that has
been evaluated in the treatment of hepatitis B, though its
35 use is associated with serious neurological side effects
~Lok, A.S., Wilson, L.A. et al., J. Antimicrob. Chemother.
(1984) 14: 93-9; Hoffnagel, J.H. et al., J. Hepatol. (1986)
W093/25239 ` PCT~US92~05091
21~5~
-- 8
3: S73-80]. ARA-AMP conjugated to arabinogalactan, and
targeted to asialoglycoprotein receptor possessing cells
where viral replication is ongoing thepatocytes), is
expected to reduce unwanted side effects by reducing the
5 concentration of the drug in the central nervous system and
increasing the concentration of the drug in the organ of
viral replication. ARA-AMP has been coupled to a
glycoprotein recognized by the asialoglycoprotein receptor
~US 4794170]. Other antiviral therapeutic agents which may
10 be used for this purpose include acyclovir and Ara-A~
A second type of anti-viral agent that can be targeted
with the teachings of the invention are antibodies. In
this context antibodies may include polyclonal antibodies,
monoclonal antibodies or antibody fragments. The natural
15 occurrence of antibodies in serum reflect past exposure to a
virus but may have little or no protective activity because
viral replication occurs within the cytoplasm of cells [I.M.
Roitt, "Essential Immunology," Blackwell Scientific, London
(l991), p. 28]. In particular hepatitis B virus replication
20 occurs within the hepatocytes of the liver and antibodies to
viral antigens cannot directly bind the virus during this
replication. If an antibody to a hepatitis B viral protein
is conjugated to arabinogalactan, it will be targeted via
the asialoglycoprotein receptor to the cytoplasm of
25 hepatocytes. Within the cytoplasm the antiviral antibody
will bind replicating hepatitis B virus and become an
effective therapeutic agent.
Some of the arabinogalactan derivatives described have
~, no known pharmacological activity, other than their ability
30 to bind the receptor, but provide a substrate for attaching
therapeutic agents, e.g., attachment to the amino, carboxyl,
sulfhydryl, phosphoryl or other functional groups of the
derivative. The resulting conjugate will target the
therapeutic agent to cells possessing the asialoglycoprotein
35 receptor, principally the hepatocytes of the liver. The
carboxyl groups afforded by the succinyl-arabinogalactan,
glutaryl-arabinogalactan and DTPA-arabinogalactan conjugates
W093/2~239 213 S 2 n ~ PCT/US92/05091
(Examples 10-12) can be used to attach molecules through the
use of carboniimides or other agents. The amino groups
afforded by the arabinogalactan hydrazide (Example 3) or
poly-L-lysine arabinogalactan (Examples 6, 8) can also be
5 used to attach therapeutic agents by a variety of reactions.
The strong positive charge of poly-L-lysine can cause some
agents such as negatively charged nucleic acids to adhere by
ionic exchange forces [Wu, G.Y. and Wu, C.H., J. Biol Chem.
~1987) 262: 4429-2232]. A preferred embodiment of this
invention is a composition comprising arabinogalactan and
poly-L-lysine, wherein the intended use is as a carrier for
genes or antisense oligonucleotides used in parenteral
~ administration ~Degols, G., Leonetti, J.P., Gagnor, C.,
Lemaitre, M., Lebleu, B., Nucleic Acids Res (1989) 17: 9341-
15 50]. In addition to poly-L-Lysine, other polymeric
molecules, such as dextrin, dextran, or albumin may be
coupled to arabinogalactan.
In another embodiment, galactose oxidase treatment of
arabinogalactan can be used to create aldehyde groups. The
20 aldehyde groups can be reacted with diamino compounds (e.g.
ethylenediamine), to form a Schiff base, followed by
reduction with sodium borohydride. The resulting amino
derivative of arabinogalactan can then be used for the
attachment of therapeutic agents.
Similarly WR2721 has been the subject of recent
- clinical studies to ascertain whether it can be used to
protect the normal cells of cancer patients during
radiotherapy [Kligerman, M.M., Liu, T., Liu, Y., He, S.,
Zhang, Z., 7th International Conference on Chemical
30 Modifiers of Cancer Treatment (1991), Clearwater, FA 338-
340] or chemotherapy [Schein, P.S, International Conference
on Chemical Modifiers of Cancer Treatment (1991), -
Clearwater, FA 341-342]. The utility of WR2721 as a
chemoprotectant has been objected to based on the lack of
35 evidence that it selectively protects normal cells; i.e. it
may protect normal and cancer cells from radiation ~The Pink
Sheet, Feb 3, 1992, 54, ~5]. The attachment of WR2721 to
W093/25239 PCTtUS92/05091
~13 .~ 2 ~ ~
-- 10 --
arabinogalactan will overcome this shortcoming, directing
the agent to cells possessing the asialoglycoprotein
receptors. The radioprotective activity of WR2721 will be
targeted to normal cells since the asialoglycoprotein
5 receptor is found chiefly on non-cancerous hepatocytes, see
above.
Free radical scavengers other than WR2721 can be
attached to arabinogalactan, and targeted to receptor
bearing cells. These scavengers include melanins ~Hill,
10 H.Z., Huselton, C., Pilas, B., Hill, G.J.; Pigment Cell Res
(1987) 1: 81-6], Trolox [Wu, T.W., Hashimoto, N., Au, J.X.,
Wu, J., Mickle, D.A., Carey, D~, Hepatology (1991) 13: 575-
~ 80~, cysteamine derivatives [Schor, N.F., Siuda, J.F.,Lomis, T.J., Cheng, B., Biochem J (1990) 267: 291-6],
15 cationic aminothiols generally, glutathiols, and vitamin E
derivatives.
After synthesis, the interaction of the arabinogalactan
derivative with the asialoglycoprotein receptor can be
determined in vivo. The ability of a derivative to interact
20 with the asialoglycoprotein receptor is assessed by its
ability to block the clearance of a substance recognized to
~ interact with the asiàloglycoprotein receptor based on
--~ earlier work. An arabinogalactan coated superparamagnetic
~ iron oxide colloid interacts with this receptor and a
~ ~ .
25 quantitative assay for its clearance has been described
below. In the absence of a blocking agent, the
arabinogalactan coated superparamagnetic iron oxide is
rapidly cleared via the asialoglycoprotein receptor with a
blood half-life of 2.8 minutes. The interaction of free
30 arabinogalactan with the asialoglycoprotein receptor effects
an increase in blood half-life of this substance, providing
a basis for evaluating the blocking ability of
arabinogalactan derivatives.
To obtain the blood half-life a Sprague-Dawley rat
(200-300 grams) is anesthetized (lO0 mg/kg of Inactin) and
injected with a defined dose of a blocking agent, followed
by an arabinogalactan coated superparamagnetic iron oxide at
W093t25239 - 2 1 3 5 ~ 9 - PCT/USg2/05091
40 umoles Fe/kg. Blood is withdrawn and 1/T1, the spin-spin
relaxation rate, determined. The enhancement in 1/T1 is
directly proportional to the concentration of
superparamagnetic iron oxide, and from changes in 1/Tl the
5 blood half-life is determined as described [Josephson et al.
Mag Res. Imag. (1990) 8: 637-646].
Table 1 indicates that arabinogalactan can tolerate a
substantial degree of modification produced by many
different types of reactions, without losing its activity as
10 a blocking agent (receptor binding activity). With
antibodies and enzymes, covalent modification especially
high levels of covalent modification, generally decreases or
- destroys biological function. Thus it i~ surprising that
arabinogalactan tolerates random modification with excellent
15 retention of its receptor-recognizing biological activity.
In fact two derivatives tested, the phosphoryl
arabinogalactan and succinyl-arabinogalactan, were more
potent as blocking agents than the parent arabinogalactan.
The basis for this highly surprising improved reactivity is
20 unknown. In contrast, lactose, a disaccharide-containing
galactose, is substantially less active a blocker than
arabinogalactan.
~-~` The ability of a derivatization procedure to damage the
binding affinity of arabinogalactan for the
25 asialoglycoprotein receptor is shown by example 18. The
~ acetate derivative has greater than 5 milli-equivalents of
- acetate per gram of arabinogalactan acetate and exhibited
substantially reduced blocking activity.
If an arabinogalactan conjugate is inactive in the
30 blocking assay, i.e., does not prolong blood half-life,
conditions used in conjugate synthesis can be adjusted to
achieve a lower degree of modification. Alternatively, the $
modification strategy employed may be dropped altogether and
a different procedure employed.
W093/25239 PC~/US92/05091
~1352~
- 12 -
TABLE 1
Interactions of arabinogalactan deri~atives
with the asialoglycoprotei~ receptor
Agent Dose Blocking Agent Half-life
(mg/kg) (min)
, _ _
None none 2.8
. _ _.
lactose 300 8.0
. ,. ~
arabinogalactan 150 33.2
phosphoryl- 150 51.0
arabinogalactan .
(Example 9)
. .
succinyl 150 213
arabinogalactan
(Example 11)
_
arabinogalactan-AMP 15Q ~100
_ (Example 4)
I
arabinogalactan- 150 86
WR2721 (Example 15)
I _
arabinogalactan 150 7.3
acetate
(Example 18)
. .
arabinogalactan 150 40.8
propionate
(Example 19)
_ _ . ,.
The examples below demonstrate that arabinogalactan can
be modified by the addition of phosphoryl, sulfhydryl,
amino, carboxyl, halo, or acylimidazol groups, with receptor
30 binding activity being unaffected. The initial modification
is performed on the hydroxyl groups on the arabinogalactan.
~- W093/25239 21~ S 2 ~J - PCT/US92/05091
- 13 -
The derivatives can be used to prepare conjugates with
therapeutic agents, as for example arabinogalactan-WR2721 or '
arabinogalactan-AMP (Table 1). In some cases we describe
the preparation of amino or carboxy arabinogalactan
5 derivatives with no known therapeutic activity. These
derivatives can be used to attach a wide range of drugs or
ligands to the amino or carboxy groups of derivatized
arabinogalactan, with generally known crosslinking and
conjugation chemistries. These derivatives can also be used
10 to attach macromolecules like genes, proteins, antibodies
and enzymes to arabinogalactan. A recent compendium of
applicable reactions is S.W. Wong, "Chemistry of Protein
Conjugation and Cross-linking," CRC Press Boca Raton, l991).
Reagents used to couple proteins to solid phase amino or
15 carboxyl groups can also be used after minor modifications
(see I. Chibata, "Immobilized Enzymes," Halstead Press, New
York 1978). Some examples of therapeutic agents that can be
conjugated to àrabinogalactan to provide useful
pharmaceutical agents are listed in Table 2.
_
r
W093~2~239 ~ PCT/US92/05091 ,~
2 9 S
- 14 -
TABLE 2
Therapeutic agents that have been or might be attached
to ara~inogalactan or arabinogalactan derivatives
, _ _ _ _ ~
Agent Class Reference
I . . ,_
Ara-AMP antiviral activity Example 5
_ _ _
WR2721 chemo- and Example 14
radioprotective
activity
.
Pepstatin anti-inflammatory Example 17
activity
: lO gene therapy Examples 6, 8
DNA .
,~ _ .
Antisense antiviral activity Examples 6, 8
nucleic acid
: Antibody to antiviral activity
: 15 hepatitis
.~
. ~ Steriod
~: anti-inflammatory
activity
I _ _ _ _
Superoxide dismutase
anti-inflammatory
¦ activity _
t
Examples
2S '~
Ex~mple 1: Bromination of arabinogalactan.
The arabinogalactan (AG) used is from the Western Larch
and chromatographs produre a single peak of about 20,000
daltons by size exclusion chromatography.
Ten grams of arabinogalactan are dissolved in 35 ml of
- W093/25239 ~1 3 5 2 ~ ~ PCT/US92/0~091
- 15 -
a 7.1% (w/v) solution of Zn(BF4)2. Fifty ml of
epibromohydrin is added and the solution stirred for so
minutes at 100C. The bromo-arabinogalactan is precipitated
in 150 ml cold (4-C) acetone, redissolved in water, and
5 precipitated in 150 ml cold ethanol. Analysis of the
product for bromine showed 0.7 milliequivalents bromide per
gram product.
Example 2: Treatment of arabinogala~tan with sodium
borohydr~e.
Ten grams sodium borohydride is added to 3,500 grams of
a 28.6% (w/w) solution of arabinogalactan. The mixture is
~ stirred overnight, and then dialyzed for six days against 35
liters of water (changing the water daily) using 3,500
dalton cut-off dialysis tubing to remove unreacted NaBH4.
The 3-methyl-2-benzothiazolone hydrazone test for
aldehyde is used to compare the aldehyde content of the
arabinogalactan starting material to sodium borohydride
reduced arabinogalactan. Arabinogalactan showed the blue
dye formation characteristic of aldehyde, reduced
20 arabinogalactan produced no dye, indicating essentially
complete reduction.
Example 3: ~ydrazino-arabinogalactan.
. .
Ten grams of reduced arabinogalactan (Example 2~ is
dissolved in 35 ml of a 7.1% (w/v) aqueous solution of
25 Zn(BF~)2. Fifty ml of epibromohydrin is added and the
solution stirred for 90 minutes at lOO C. The brominated
arabinogalactan is precipitated in 150 ml cold (4-C~
acetone, redissolved in water, and precipitated in 150 ml
cold ethanol. Five grams of this brominated arabinogalactan
30 product is dissolved in 15 ml of 0.3 M aqueous borate, pH 8.
Ten grams hydrazine is added and the mixture is stirred for ~:
24 hours at room temperature. The hydrazido-arabinogalactan ^~
is precipitated in 150 ml cold (4-C) acetone, redissolved in
water and precipitated in 150 ml cold ethanol. The product
35 hydrazide content is analyzed by acid-base titration and
~howed 0~25 milliequivalents hydrazide per gram of product.
W O 93/25239 PC~r/US92/05091 t -
21352~5
- 16 -
Ex~mple 4: Arabino~alactan conjugated to adenosine
5~ monopho~phate ~AMP)
One gram (2.9 mmoles) of adenosine 5'-monophosphate
(AMP) is dissolved in 20 ml water with the addition of !-
5 sodium bicarbonate powder. Arabinogalactan-hydrazide (0.6
g, example 2) is added and the pH adjusted to 7.5 with
sodium hydroxide. One gram (5.2 mmoles) of 1-ethyl-3,4-
- dimethylaminopropyl) carbodiimide is added and the reaction
maintained at room temperature for 64 hours. The product is
10 purified by ultrafiltration using an Amicon YM3 ultrafilter,
further purified by precipitation from ethanol. A yield of
- 323 mg of product was obtained. The product was analyzed by
cation exchange chromatography (Rainin Synchropak, strong
cation exchange So 300 A, 25 x 0.5 cm column) using a
15 buffer of 0.1 mM, pH 7.0 phosphate buffer at flow rate o.5
ml/min). A single broad pea~ at 5.7 minutes with no
evidence for underivatized AMP ~retention time 6.3 minutes)
was observed. The number of AMP molecules per gram of ~G-
AMP product, based on the comparison of HPLC area under the
20 curve monitoring at 260 nm is 0.24, indicating approximately
a 95% conversion of available hydrazide groups. The UV/VlS
, ,
spectrum of the AG-AMP product is virtually identical to
underivatized AMP. The analysis of product by size
~ exclusion (Amicon Cellufine GC200M) chromatography shows a
-~ 25 molecular weight approximately equivalent to underivatized
arabinoga~lactan, about 20,000 daltons.
The activity of AG-AMP was evaluated in the animal
model as described above. 150 mg/kg of this substance was
an effective blocker of the superparamagnetic iron-oxide
30 colloid, extending the half-life of the colloid to greater
than 100 minutes (Table 1).
Ex~mple 5: Arabinogalactan conjugated to adenine 7
ar~binoside s~ monopho~phate (ARA-AMP).
One gram (2.9 mmoles) of adenine arabinoside 5'-
35 monophosphate (ARA-AMP) is dissolved in 20 ml water with the
addition of sodium bicarbonate powder. 0.6 grams of
arabinogalactan-hydrazide (Example 2 above) is then added
'
.
W093~25239 ~1 352~ - PCT/US92/0509l
and the pH adjusted to 7.5 with the addition of sodium
hydroxide. One gram (5.2 mg) of 1-ethyl-3,4-
dimethylaminopropyl) carbodiimide is added and the reaction
maintained at room temperature ~or 64 hours. The product is
5 purif~ed by ultrafiltration using an Amicon YM3 ultrafilter,
and then further purified by precipitation fram ethanol and
dried, yielding 280 mg of product. As with AG-AMP (Example
3), the strong cation exchange chromatography showed a
single broad peak centered at 5.7 minutes and no measurable
10 residual unreacted ARA-AMP. The UV/VIS spectrum of AG-ARA-
AMP was ~irtually identical to an ARA-AMP standard. Based
_ on the area under the curve at an optical density of 260
nanometers and in comparison with an AMP standard, which is
assumed to have the same extinction coefficient as ARA-AMP,
15 the product has 0.124 milli-equivalents of ARA-AMP per gram.
Ex~mple 6: Poly~L)lysyl-~rabinog~la~t~n ~prep~re~ by
re~uctive ~mination).
Poly~L)lysine hydrochloride (1,000-4,000 daltons, 0.5
grams) is dissolved in 2 ml borate buffer (0.2M) and the pH
20 adjusted to 9.0 with sodium hydroxide. 100 mq
arabinogalactan and 50 mg sodium cyanoborohydride is added.
~- and the reaction heated for 24 hours at 50-C. The product
mixture is puriied using an Amicon YM3 ultrafilter. The
retentate containing the polylysine-arabinogalactan
25 conjugate showed a positive ninhydrin test for amine and
positive anthrone test for polysaccharide. The yield was 30
mg. Si~e exclusion high performance liquid chromatography
(Amicon Cellufine GC200M) showed a product having a
molecular weight approximately equal to the sum of molecular
30 weights of arabinogalactan and poly~L)lysine or about 25,000
daltons.
Ex~mple 7: Acylimidazole-arabinogalact~n.
Three grams of anhydrous arabinogalactan is suspended
in 5 ml of anhydrous peroxide free dioxane. While stirring,
35 1.62 gm (lOmmole) of N,N'-carbonyl diimidazole, dissolved in
10 ml of dioxane, is added in a single portion. After
stirring for 20 minutes the acylimidazol-arabinogalactan is
W093/25~39 PCT/US92/05U91 ,~~
- ~8 -
collected by fil~ration (medium frit). The praduct is
washed with 25 ml of dioxane and refiltered. A second
dioxane titration is performed. The product is next
titrated with 25 ml of apf-diethyl ether and then vacuum
5 dried. Yield is 2.9 gm.
Ex~mple 8: Poly~L)ly~ine-arabi~ogalactan ~prepared rom
acylimidazole-arabinogalactan).
One gram of acylimidazole-arabinogalactan (Example 7
and 0.2 grams poly~L)lysine (1,000-4,000 daltons) is
10 dissolved in 5 ml of 0.2M borate buffer and the pH adjusted
to 8.6 with sodium hydroxide. The reaction is allowed to
- proceed for 24 hours at 5C. The product is isolated first
by precipitation in ethanol, and then purified using an
Amicon YM10 ultrafilter. The retentate shows a positive
15 test for amine and carbohydrate using the ninhydrin and
anthrone tests, respectively, while the final filtrate is
negative for amine. The yield is 310 mg.
The product poly(L)lysyl-arabinogalactan is analyzed by
cation exchange chromatography HPLC (Rainin Synchropak
20 strong cation exchange resin, So 300A, 25 X 0.5 cm), using
pH 5.5, 25 mM phosphate buffer at a 1 ml/min flow rate.
The product, arabinogalactan-polylysine, elutes with a
retention time of 3.9 minutes whereas unconjugated
polylysine elutes with a retention time of 9.3 minutes.
25 Poly(L)lysine bound to arabinogalactan is verified by its UV
speatrum.
Example 9: Phosphoryl-arabinogalactan.
Two grams of arabinogalactan are dissolved in 20 ml
formamide and 4 ml triethylamine. Ten grams polyphosphoric
30 acid are added and the reaction stirred for 1~ hours. The
product is brought to pH 9 with 45~ NaOH and ultrafiltered
in a SO ml stirred cell with a 3,000 molecular weight cutoff
membrane ~Amicon), bringing the volume from 50 ml to 10 ml
twice. The ultrafiltered product is precipitated into 500
35 ml cold acetone (4-C), redissolved, and precipitated in SOO
ml cold ethanol. The product showed 0.21 milli-equivalents
of phosphate per gram of product both by acid base titration
W093t25239 _ 2 1 3 5 2 ~ ;~ PCTiUS92/05091
-- lg --
and by colorimetric quantitation of inorganic phosphate
(inorganic phosphorus kit, Sigma Chemical, St.Louis, MO)
following trifluoroacetic acid hydrolysis (2M acid for 1
hour at 120-C).
The activity of phosphorylated arabinogalactan was
evaluated in the animal model as described above. 150 mg/kg
of this substance was an effective blocker of the
superparamagnetic iron-oxide colloid, extending the half-
life of the colloid to greater than 51 minutes (Table 1).
10 Example 10: Treatment of arabinogalactan with galactose
oxida~e (GO).
- Ten grams of arabinogalactan is dissolved to a total
volume of about 50 ml in O.1 M potassium phosphate buffer,
pH = 6Ø To the resultant solution is added 225 units of
15 galactose oxidase dissolved in about 2 ml of the same
buffer. The oxidation is allowed to proceed for 24 hours at
room temperature. The H2O2 content is found to be about 3
mg/ml, as measured by peroxide test strips. A second
addition of 22S units of GO is made to the reaction mixture.
20 After another 24 hour reaction period the peroxide content
is found to be unchanged from the result of the first GO
treatment at about 3 mg/ml. Twenty milligrams of catalase ~--
(dry solid) is added to decompose the peroxide. After
standing at room temperature overnight the contents of the
-- 25 flask are found to be free of peroxide. }
Product Purification. Ten grams of mixed bed resin,
MB-l is added to the flask. After stirring for 30 minutes
the solution is decanted into and passed through a short
column containing an additional 5 grams of MB-l resin. The
30 pH neutral solution is found to be free of any protein
amines by reaction with ninhydrin. The product is isolated
by precipitation from 5-C cooled absolute ethanol. The
precipitate is collected by filtration. The aldehyde
content of this product is found to be between 3 and 5 times
35 greater than the aldehyde content of native arabinogalactan.
Yield is lO grams.
Determination of the Number of Aldehyde Groups: The 3-
--
W093/25239 PCT/U~92/0509l ~
~13~
- 20 -
methyl-2-benzothiazolone hydrazone test for aldehyde was
used to compare arabinogalactan starting material to poly-
aldehydic arabinogalactan. Based on absorbance measured at
670 nm, this poly-aldehydic arabinogalactan has 0.34 milli-
5 equivalents aldehyde per gram of ara~inogalactan.Ex~mple 11: ~uGcinyl-arabinogalactan.
Purified arabinogalactan (16.0 g, 0.70 mmol) and
succinyl anhydride (10.0 g, 100 mmol) were dissolved in DMSO
(200 ml) at 60-C. After 1.0 hour, the clear, light yellow
10 solution was cooled to ambient temperature and allowed to
stir for an additional 48 h. The DMSO solution was added to
- H2O (200 ml), filtered on an Amicon YM3 ultrafiltration
membrane and washed with H2O (3 times with 250 ml). The
solution remaining on the membrane was frozen and
15 lyophilized. Yield of white powder: 20.~ g. IR (KBr):
1732 cm1 (C=O). Titration of an aqueous solution of the
conjugate with G.01 N NaOH indicated the presence of 1.96
milli-equivalents succinate per gram of succinyl-
arabinogalactan.
The activity of succinyl-arabinogalactan is evaluated
in the animal model as described above. 150 mg/kg of this
substance was an effective blocker of the superparamagnetic
iron-oxide colloid, extending the half-life of the colloid
to 213 minutes (Table 1).
25 Example 12: DTPA-arabinogalactan.
Purified arabinogalactan (20.0 g, 0.87 mmol) and the
dianhydride of diethylenetriaminepentaacetic acid (DTPA)
(2.15 g, 6.02 mmol~ were dissolved in dimethylsulfoxide
(DMSO, 200 ml) at 60 C. After 0.5 hour, the clear solution
30 was added to HzO (ca. 500 ml) at 15 C. The solution was
filtered on an Amicon YM3 and YMl ultrafiltration membranes
t5,000 and 1,000 dalton cutoff, respectively) and washed
with H2O (2 X 400 ml). The solution (70 ml) remaining on
the membran~ was frozen and lyophilized~ Yield of white
35 powder was 18.8 g. The IR showed a band at 1734 cm~l (C=O).
Titration of an aqueous solution of the conjugate with 0.010
N NaOH indicated the presence of 0.117 milliequivalents DTPA
Wo 93/2~239 2 13 ~ ~ ~ r? PCT/US92/0~091 1
-- 2 1
per gram DTPA-arabinogalactan.
Example 13: Gluta~yl-arabinogalactan.
Purified arabinogalactan (20.0 g, 0.87 mmol) and
glutaric anhydride ~5.00 grams, 44 mm~l~ were dissolved in
5 DMSO (200 ml) at 60 C. The reaction mixture was cooled to
ambient tempera~ure and allowed to react for 16 hours. The
DMSO solution was added to H2O (200 ml), filtered on an
Amicon YM3 ultrafiltration membrane and washed with H20 (2
times 300 ml). The solution remaining on the membrane was
frozen and lyophilized. Yield of white powder: 18.5 g (lot
number 2127-17~). IR (KBr): 1726 cm~~ (C=o).
- Example 14: S-2-t3 aminopropylamino) ethyl-thiopho~phate-
dextran-arabinogalactan from thiopho~phorylated dextran.
PolythioPhos~horYlation of_dextran. Tén grams of :
15 dextran is suspended in 60 ml of anhydrous pyridine. The
suspension is cooled in an ice water bath~ To the cooled
suspension is added dropwise with stirring 10 ml (98.4
mmoles) of thiophosphoryl chloride. Once the addition is
complete the reaction mixture is allowed to warm to room
20 temperature with constant stirring. The reaction flask is
then immersed in an oil bath and heated for 16 hours at
40-C. ,,
The slightly yellow colored reaction mixture is cooled
in an ice bath. Once cooled, water is`added slowly dropwise
25 while the reaction suspension is vigorously stirred. After
about 10 ml of water has been added to the reaction mixture
a solution of 1 N NaOH is added until a pH of 9.5 is
reached. The solution is then evaporated at room
temperature to an oil. The residue is mixed with 20 ml of
30 water, which results in a clear homogeneous solution. This
solution is added dropwise to 200 ml of 0 C ethanol which is
vigorously stirred. The resulting white precipitate is
collected on a coarse fritted funnel and dried under vacuum.
Titration with 0.5 M hydrochloric acid indicates that 1
35 mmole of thiophosphate is incorporated per gram of
polysaccharide.
Svnthesis of 2-f3-aminoproPYlamino) ethvl bromide,
W093~25239 PCT/US92/0509l
213~29~
- 22 -
dihydrobromide. Twenty three and six-tenths grams (200
mmole) of ice cold ~-(w-aminopropylamino) ethanol is added
portionwise to 200 ml o~ ice cold 48-52% hydrobromic acid.
After stirring for 1 hour the reaction mixture is heated to
5 reflux for 1~-20 hours. The reaction mixture is vacuum
dried to a reddish colored oil. The oil is titrated with
300 ml of acetone and left under refrigeration for 4 hours.
The mother liquor of acetone is decanted away from the gummy
residue. The residue is dissolved with 75 ml of water and
lO the resulting solution is added to 600 ml of cold acetone.
The crystalline precipitate is collected and then dissolved
_ in boiling methanol. The resulting methanol solution is
added to a ~0% mixture of ethyl ether and acetone (400 ml).
After cooling the mixture overnight the pure white crystals
15 are collected and vacuum dried. The melting point of the
product is 205-206-C, as reported [Piper, J.R., et.al.
(1969), J. Med. Chem 12: 236-~43].
Reaction of polvthio~hosphorYlated dextran with 2-(w-
aminopr~ylamino) ethyl bromide to form S-2-(3
20 aminopropylamino) ethyl-thiophosphate-dextran. Five mmoles
of polythiophosphorylated dextran, sodium salt, is dissolved
in lO ml of water. To the above solution is added 5.5
mmoles of 2-(3-aminopropylamino) ethyl bromide
dihydrobromide dissolved in 10 mls of water. The clear
25 solution is stirred for four hours at room temperature. The
resultinq turbid solution is added dropwise to rapidly
stirred O-C ethanol. The resulting precipitate is collected
by filtration. The product is washed with twice with 25 ml
portions of warm (40-50-C) ethanol and vacuum dried.
The extent of thioalkylation is determined by a
colorimetric analysis with ninhydrin.
Reaction of S-2-(3 aminopropYlamino) ethyl-
thiophosphate-dextran with Arabinoqalactan-acylimidazole
S-2-(3 aminopropylamino) ethyl-thiophosphate-dextran is
35 reacted with arabinogalactan-acylimidazole (Example 7) at
4-C for 16 hours. The product is isolated and purified by
ultrafiltration using a YMlO filtration membrane.
-`~` W093/25239 2 1 3 ~ ~ 9 5 PCT/US92/05091
- 23 -
Example 15: Arabinogalactan-WR2721 from brominated
nrabinogal~ctan.
Reduced arabinogalactan is bro~inated as described in
Example 3. 2 grams of this brominated arabinogalactan is
5 added to 1 gram of WR2721 in 10 ml of 0.2M borate and the pH
adjusted to 8Ø The mixture is stirred for 16 hours at
room temperature. Arabinogalactan-WR2721 is purified by
Amicon YM3 ultrafiltration, then precipitated in acetone and
redissolved in water. Finally it is precipitated in ethanol
10 and dried. The final product is dissolved in 0.1 N HCl and
titrated with 0.1 N NaOH. Using WR2721 as a reference for
- the titration, the arabinogalactan-WR2721 final product was
shown to have 0.66 milli-equivalents of WR2721 per gram of
product. The product analyzed by size exclusion
lS chromatography ~Amicon Cellufine GC200M) shows the major
component has a molecul~r weight of about 25,000 daltons.
The activity of arabinogalactan-WR2721 was evaluated in
the animal model as described above. Injection of 150 mg/kg
of this substance was an effective blocker of the
20 superparamagnetic iron-oxide colloid clearance, extending
~ the half-life of the colloid to 86 minutes (Table 1).
Ex~mple 16: Arabinogalactan-WR2721 from pho~phorylated
arabinogalactan.
Arabinogalactan-phosphate (8 grams, example 9), 1.2
25 grams 1-ethyl -(3,4-dimethylaminopropyl~carbodiimide, and 1
gram of WR2721 are mixed together in 20 ml of water. The pH
is adjusted to 7.5 with the addition of sodium hydroxide,
and the mixture allowed to stand in the dark at room
temperature for approximately 64 hour~. The product, WR2721
linked to arabinogalactan through its primary amine
esterified to the phosphate on arabinogalactan-phosphate, is
purified by ultrafiltration (5 times 10 ml) using a YM3
(3000 daltons cutoff) and then freeze dried. The yield is
0.63 grams of white crystalline powder.
35 Characterization:
i. Molecular weight. Size exclusion chromatography
(Amicon Cellufine GC200M) showed a single peak centered at
W~93/25239 PCT/US92/05091 -~
213~2~5
- 24 -
22 minutes, similar to that observed for arabinogalactan-
phosphate starting material. No evidence was seen of 1DW
molecular weight impurities.
ii. Analysis of sulfhydryl content. The product phosphate
linked arabinogalactan-WR2721 is first hydrolyzed in 2M
trifluoroacetic acid for 1 hour at 120C. After
neutralîzation, the sulfhydryl concPntration is measured by
a colorimetric test using 5,5' bisdithio 2-nitrobenzoic
acid. The amount of WR2721 on arabinogalactan was
lO determined to be 0.063 milli-equivalents per gram of
product.
- iii. Enzyme catalyzed hydrolysis. Both alkaline
phosphatase (Biozyme Code ALPI-12G) at pH 8.0 and acid
phosphatase (EC3.1.3.2, from potato) at pH 4.8 were found to
15 rapidly hydrolyze the phosphothioate ester and thus unblock
the thiol. The rate of hydrolysis by the acid phosphatase
was 0.1 micro-equi~alents phosphate/minute at 27-C, a rate
which is close to that expected from the hydrolysis of p-
nitrophenyl phosphate.
20 Example 17: Arabinogalactan-pep~tatin.
Pepstatin can be conjugated to amino-arabinogalactan
(2% amine by weight polysaccharide) through a N-hydroxy
succinimide ester ~Furuno, K., et.al. (1983) J. Biochem 93:
249~. Arabinogalactan with a primary amine is prepared
25 according to Example 2 (arabinogalactan-hydrazide) or
example 5 or 7 (polylysine-arabinogalactan). Dissolve
pepstatin A (250 mg) in 1 ml of dimethylformamide. Then add
50 mg 1-ethyl-3(3-dimethyl-aminopropyl)carbodiimide and 30
mg of N-hydroxy succimide. After the reaction has proceeded
30 at room temperature for 2 hours, add the mixture dropwise to
30 ml of 0.1 M sodium bicarbonate containing lOO mg of
amino-arabinogalactan. Allow the resultant mixture is sit
at room temperature for 2 h, then purify the product by
ultrafiltration using a 10,000 dalton cutoff, and then by
35 cationic exchange chromatography.
-~ W093J2~239 ~1 3 5 2 ~ ~ PCT/US92/05091
- 25 -
Example 18: Carboxymethyl-arabinogalactan from reaction of
bromo~cetic ~cid with arabinog~lactan
Five grams of arabinogalactan is dissolved in 50 ml of
4N sodium hydroxide. To this is added lo grams of
5 bromoacetic acid, and the mixture heated at 80 D c for three
hours. The reaction is terminated by cooling to room
temperature then adjusting the pH to between 7.5 and 9 using
concentrated hydrochloric acid. The product is isolated and
purified by G-25 column chromatography and ultrafiltration
10 using an Amicon YM3 membrane. The extent of derivatization,
ascertained by running the reaction with 14C labeled
^ bromoacetic acid and measuring the specific activity of the
product by liquid scintillation counting, is about 5.2
milli-equivalents of carboxymethyl groups per gram of
15 product.
The activity of this arabinogalactan acetate was
evaluated in the animal model as described above. A dose of
lSO mg~kg was not an effective blocker of the
superparamagnetic iron-oxide colloid, extending the half-
20 life of the colloid only to 7.3 minutes, compared to 33.2minutes for underivatized arabinogalactan (Table 1).
Example 19: Carboxyethyl-arabinogalactan from reaction of
2-bromopropionic acid with arabinogalactan
Fi~e grams of arabinogalactan is dissolved in 50 ml of
25 4N sodium NaOH. To this is added 11 grams of 2-
bromopropionic acid, and the mixture heated at 80C for
three hours. The reaction is terminated by cooling to room
temperature, then adjusting the pH to between 7.5 and 9
using concentrated hydrochloric acid. The product is
30 isolated and purified by G-25 column chromatography and ;
ultrafiltration using an Amicon YM3 membrane. The extent of ~-
derivatization, determined by acid/base titration, is about
1.3 milliequivalents propionate per gram of product. ;'
The activity of arabinogalactan propionate was
35 evaluated in the animal model as described above. Use of
150 mg/kg of this substance showed it ineffective blocker in
superparamagnetic iron-oxide colloid clearance assay,
W093/25239 ~ PCT/US92~0~091 ~
~1352~S - ~6 -
extending the half-life of the colloid to 40.8 minutes
(Table 1).
Example 20: Arabinogalactan-W~2721 from thiopho~phorylated
arnbinogal~ctan
Thiophosphorylatlon of arabinoqalactan. Ten grams of
anhydrous arabinogalactan is suspended in 50 ml of
triethylphosphate. After the addition of 10.5 ml ~75
millimole) of anhydrous triethyl amine, the suspension is
cooled in an ice-water ~ath. To the cooled suspension is
10 added dropwise with stirring 2.55 ml (25 millimole) of
thiophosphoryl chloride. Once the addition is complete, the
- reaction mixture is warmed to room temperature and stirred
for 72 hours. After this time, the arabinogalactanyl
thiophosphorodichloridate product is hydrolyzed by adding 50
15 ml of deionized ice-water and stirring for two hours. The
solvent, triethyl phosphate, is removed from the reaction
mixture by extraction with 2 times with 25 ml portions of
chloroform. The pH of the aqueous phase is adjusted to
between g and 9.5 by the addition of 1 N sodium hydroxide.
20 The product is purified by ultra-filtration (50 ml to lO ml,
~- four cycles) using an Amicon YM3 (3000 dalton cutoff)
ultrafiltration membrane. The final retentate is
lyophilized to dryness.
Synthesis of 2-(3-aminopropylamino) eth~l bromide.
25 dihydrobromide. The synthesis of 2-(3-aminopropylamino)
ethyl bromide, dihydrobromide is as described in Example 14~
Reaction of polythiophosPhorylated arabinoqalactan with
2-(w-aminopropylamino) ethvl bromide to form S-2-(3
aminopropylamino) ethvl-thio~hos~hate-arabinocalactan. Five
30 mmole of polythiophosphorylated arabinogalactan, sodium
salt, is dissolved in 10 ml of water. To the above solution
is added 5.5 mmole of 2-(3-aminopropylamino) ethyl bromide
dihydrobromide dissolved in 10 ml of water. The clear
solution is stirred for four hours at room temperature. The
35 resulting turbid solution is added dropwise to rapidly
stirred O-C ethanol. The resulting precipitate is collected
by filtration. The product is washed twice with 25 ml
,~ W093/25239 213 5 ~ 9 5 PCT/US92/05091
- 27 -
portions of warm (40-50 C) ethanol and vacuum dried.
Thioalkylation is confirmed by a colorimetric analysis
with ninhydrin.
