Note: Descriptions are shown in the official language in which they were submitted.
WO 95/09637 2 1 ~ ~ ~ 6~ PCT/US94111368
.
HIGHLY SULFATED MALTOOLIGOSACCHARIDES
WITH HEPARIN-LIKE PROPERTIES
Field of the Invention
5The invention relates to a novel class of highly sulfated
maltooligosaccharides having heparin-like activity, and to methods for using these
oligosaccharides, alone or in combination with a chemotherapeutic drug, to treatcertain diseases.
10 Backaround of the Invention
Heparin and other naturally occurring glycosaminoglycans, such as dermatan
sulfate or heparan sulfate, possess medically useful properties. Recently, several
low molecular weight heparins from various chemical or enzymatic depolymerization
processes have been developed for clinical use. Thomas et al., Thrombos. Res.
15 (1982) 28:343-350; Walenga et al., Thrombos. Res. (1986) 43:243-248; Koller et al.,
Thrombos Haemostas. (1986) 56:243-246. For instance, the involvement of heparin
or heparan sulfate or degradation products thereof in smooth muscle proliferation
has been recognized for some time. Heparin and heparan sulfate can slow or
arrest the vascular proliferation associated with injury described hereinabove
20 (Clowes, A.W., et al., Nature (1977) 265:625-626). The effect of heparan sulfate
and heparin on smooth muscle proliferation is also described by Marcum, J.A., etal. in Bioloav of Proteoalycan, Academic Press (1987) pp. 301-343. The inhibition
of vascular smooth muscle cell growth by heparin was further described by
Castellot, J.J., Jr., et al., J Biol Chem (1982) 257:11256-11260, and the effect of
25 heparin on vascular smooth muscle cell growth in fetal tissue was described by
Benitz, W.E., et al., J Cell Physiol (1986) 127:1-7. The effect of heparin as aninhibitor of both pericyte and smooth muscle cell proliferation was shown by
Orlidge, A., et al., Microvascular Research (1986) 31:41-53, and these authors
further showed that chondroitin sulfate, and dermatan sulfate do not have this
- 30 effect. A review of the effects of heparin and heparan sulfate on the proliferation of
smooth muscle cells has been published by Benitz, W.E. in "The Pulmonary
W0 95/09637 ~ 5 PCT/US94/11368
.
Circulation: Normal and Abnormal", Fishman, A.P., ed., University of Pennsylvania
Press (1988).
Heparins and certain low molecular weight heparins have certain
disadvantages for medical applications. Generally, both compositions exhibit
5 anticoagulant activity, and thus must be administered with considerable medical
supervision. Recently, however, non-anticoagulant heparins have been developed
to circumvent this problem and are now undergoing clinical trials. Secondly,
heparins are of natural origin, generally being purified from animals. As such, small
amounts of the glycosaminoglycans may cause anaphylactic reactions, a decrease
10 in the number of thrombocytes, thrombosis and embolism. Therefore, the
preparation or identification of synthetic agents possessing properties comparable to
heparin is desired.
Biologically active hexose polymers or modified heparins have been
described previously that display certain heparin properties. U.S. Patent No.
4,066,829 discloses complement system-modulating activity for sulfated
maltodextrin polymers of undisclosed molecular weight. Similar complement
system-modulating activity for 4-O-polyhexose-thio-arylene sulfate derivatives is
disclosed in U.S. Patent No. 4,470,976 and U.S. Patent No. 4,435,387. The
hexose polymers described are substituted disaccharides.
EPA 0338092 describes alkali metal or alkaline earth metal salts of a
sulfated linear polymer of 4 to 10 D-glucose units linked a-1,4. These compositions
are claimed to have anti-HlV activity.
PCT patent application, WO 92/01003, shows that certain non-anticoagulant
heparins act as heparanase inhibitors, and that they may be effective in lessening
25 or preventing lung colonization by metastatic cell variants. Two scientific a bstracts
by Fugedi et al describe sulfated maltooligosaccharides. See, XVlth International
Carbohydrate Symposium, July 5-10th, 1992, Paris-France: Modulation of bFGF
Biological Activities by Sulfated Maltooligosaccharides, page 402; and AntiviralActivity of Sulfated Maltooligosaccharides, page 446. That these compositions can
30 bind basic fibroblast growth factor and inhibit herpes simplex virus plaque formation
is also described. However, there is no description of the extent of sulfation of
WO9S/09637 2 ~ 7 ~ ~ ~ 5 PCT/US94/11368
these compositions, and what effect high levels of sulfation would have on
biological activity.
Summary of the Invention
The instant invention provides highly sulfated maltooligosaccharide
compositions having therapeutic and/or prophylactic properties alone or in
combination with a chemotherapeutic drug. In certain respects the compositions
have properties similar to heparin.
In another respect the invention provides highly sulfated
10 maltooligos~ccharide compositions that are effective inhibitors of smooth muscle
cell proliferation.
A third aspect of the invention is the description of highly sulfated
maltooligosaccharide compositions that have the following structural formula:
~ OR2 OR2
.~R20_ ,, ~ , ~XR1
oR2 oR2
~ - n
where X represents O or S;
each R' independently represents an alkyl, aryl, or aralkyl group, a reduced
or oxidized glucose unit, SO3M, or H;
R2 represents a SO3M group or H;
M represents a biologically acceptable cation; and
n represents an integer from 1 to 9;
with the proviso that at least 50% of R2 groups are SO3M.
A fourth aspect of the invention is the description of preferred highly sulfated30 maltooligosaccharide compositions including sulfated maltotetraose, sulfated
maltopentaose, and sulfated maltohexaose.
WO 95/09637 PCTIUS94/11368
~7~465
A fifth aspect of the invention is the description of sulfated
maltooligos~ccharide compositions that differentially affect cell proliferation
depending on the degree of sulfation of the compositions.
A sixth aspect of the invention is the description of highly sulfated
5 maltooligosaccharide compositions that can be beneficially applied to the treatment
or prevention of certain diseases including cancer, cardiovascular disease,
retinopathies, inflammation, and diseases of viral origin.
These and other aspects of the invention will become apparent to a
practitioner of this art upon a full consideration of the disclosure presented below.
Brief Descriplion of the Drawin~s
Figure 1 shows the effects of the degree of sulfation of certain
maltooligosaccharides on binding of RO-12 UC cells to bFGF coated microtiter
wells.
Figure 2 shows the effects of the degree of sulfation of certain
maltooligos~ccharides on the inhibition of adrenocortical endothelial cell
proliferation.
Figure 3 shows the inhibition of binding of RO-12 UC cells to bFGF coated
microtiter wells as a function of the size of certain sulfated maltooligosaccharides.
Figure 4 shows the inhibition of adrenocortical endothelial cell proliferation
as a function of the size of certain sulfated maltooligosaccharides.
Figure 5 shows the effects of highly sulfated maltotetraose on the growth of
the tumor cell line MDA-231 in nude mice.
Figure 6 shows the effects of highly sulfated maltotetraose on CAPAN-2
25 tumor cell growth in nude mice.
Figure 7 shows the effects of highly sulfated maltotetraose on the growth of
the tumor cell line, PC-3, in nude mice.
Figure 8 shows the inhibitory effects of highly sulfated maltotetraose on
smooth muscle cell growth. The effects are depicted relative to porcine mucosal
30 heparin.
Figure 9 shows the inhibitory effects of highly sulfated maltopentaose on
=
WO 95/09637 PCT/US94/11368
~ 2~71465
smooth muscle cell growth. The effects are depicted relative to porcine mucosal
heparin.
Figure 10 shows the inhibitory effects of highly sulfated maltohexaose on
smooth muscle cell growth. The effects are depicted relative to porcine mucosal
5 heparin.
Figure 11 shows the effects of highly sulfated maltotetraose on heparanase
inhibition.
Description of SPecific Embodiments
All publications and patent applications discussed or cited herein are
understood to be incorporated by reference to the same extent as if each individual
publication or patent application was specifically and individually set forth in its
entirety.
The instant invention provides compositions having therapeutic properties
15 similar to heparin. The compositions of the instant invention are highly sulfated
maltooligosaccharides having the general structure given by the formula:
-
r oR2 -- r oR2
~0 ~0
R2 o 1~ ~XR1
oR2 oR2
_ n
where X represents O or S;
each R' independently represents an alkyl, aryl or aralkyl group, a reduced
or oxidized glucose unit, SO3M or H;
R2 represents a SO3M group or H;
M represents a biologically acceptable cation; and
n represents an integer from 1 to 9;0 with the proviso that at least 50% of R2 are SO3M.Typical examples of biologically acceptable cations are alkali metals, alkaline
WO95/09637 ~'~ 7 ~ l~ 65 PCT/US94/11368
earth metals, aluminum, ammonia, zinc, and substituted ammonia wherein the
substitution may produce a di- or trialkylamine (C1 -C6), piperidine, pyrazone,
alkanolamine (C2-C6), or cycloalkylamine (C3-C6), although acceptable cations are
not limited to these. Any cation providing reasonable solubility and low or no
5 toxicity and which does not significantly, or advérsely affect the pharmaceutical
properties of the parent composition is acceptable.
Preferred highly sulfated compositions of the instant invention are
maltotetraose, maltopentaose, maltohexaose, maltoheptaose, maltooctaose,
maltononaose and maltodec~ose.
The compositions of the invention can be made by treating a
maltooligosaccharide, or a derivative thereof as described below, with a sulfating
agent in an appropriate solvent by methods which are well known in the art.
Maltooligosaccharide starting materials for the synthesis of the compositions of the
instant invention include oligosaccharides having 1 to 9 D-glucose residues linked
15 oc-1,4 or mixtures of these oligosaccharides. Derivatives of these oligosaccharides,
such as O- or S-glycosides or reduced alditol derivatives, are useful as starting
material as well. It will be apparent that the structure of the final sulfated
oligosaccharide desired will determine the nature of the starting material.
Sulfating agents useful in preparing the compositions of the instant invention
20 include but are not limited to sulfur trioxide:pyridine complex, sulfur
trioxide:trimethylamine complex, and chlorosulfonic acid. Organic solvents useful
for the preparation of the compositions of the instant invention include but are not
limited to N,N-dimethylformamide (DMF), dimethylsulfoxide, and pyridine. Using
techniques known in the art, selective sulfation of the hydroxyl groups can be
25 obtained. After sulfation, the sulfate groups can be modified to possess biologically
acceptable cations, including but not limited to Na, K, Li, Ca, Mg, NH4, Al,
ethanolamine, triethanolamine, morpholine, pyridine and piperidine.
A typical sulfation reaction is carried out by dissolving the starting material
(0.5 9) in N,N-dimethylformamide (20 mL) and adding sulfur trioxide pyridine
30 complex (2 equivalents/OH groups). The mixture is stirred at room temperature for
an appropriate time, preferably 2 days. The pH is adjusted to 9 by the addition of
WO9S/09637 2 1 7 1 4 6 ~ PCTIUS94/11368
.
1 M NaOH, then the crude product is either precipitated by the addition of an
organic solvent (as e.g. ethanol), or if this is not appropriate the mixture is
evaporated to dryness under reduced pressure. The crude product is then purified to remove inorganic salts, and finally converted into the desired salt form.
An important aspect of the instant invention is the sulfate content of the
products. The products are characterized as the sulfur content determined by
elementary analysis. Because of the hygroscopic nature of the sulfated
compositions this cannot be regarded as a reliable method, therefore in the present
work the sulfate content was determined from the carbon/sulfur ratio which is
10 independent from the moisture content of the samples. The sulfate content of the
products can be expressed as the sulfation ratio, which is the ratio of the number of
sulfated hydroxyl groups to the total number of hydroxyl groups in the starting
material, expressed as percentage.
Depending on the specific sulfation method employed, the sulfate esters
15 prepared will vary in the number and position of sulfonic acid substituents. In most
cases, a mixture of sulfate esters of the starting oligosaccharide(s) will be obtained.
Preferred compositions have about 50% of the hydroxyl groups sulfated. More
preferred are compositions having 75% or greater of the hydroxyl groups sulfated.
Mixtures of different maltooligosaccharides may be sulfated and utilized for
20 medical applications. For example, a maltooligomer mixture is commercially
available which includes linear homologs from the tetrasaccharide to the
decasaccharide. Such a mixture was highly sulfated using the conditions described
herein (0.259 maltooligomer mixture in 10ml DMF treated with sulfur trioxide
pyridine complex).
Labeled Forms of the Invention Non-Anticoaqulant Compositions
The compositions of the invention can be provided with fluorescent,
radioisotope, or enzyme labels as desired. Conventional techniques for coupling of
label to carbohydrates or related moieties can be used. Such techniques are well30 established in the art. See, for example, U.S. Patent No. 4,613,665. The labeled
mixtures of the invention may be used to identify sites of disease as well as in
WO 9S/09637 PCT/US94/11368
~7~65
competitive immunoassays, and as a means to trace the pharmacokinetics of the
compositions in vivo. Suitable radioisotope labels for this purpose include
hydrogen3, iodine131, indium111, technetium99, phosphorus32, and sulphate35. Suitable
enzymic labels include alkaline phosphatase, glucose-6-phosphate-dehydrogenase,
5 and horseradish peroxidase. Particularly preferred fluorescent labels include
fluorescein and dansyl. A wide variety of labels of all three types is known in the
art.
Administration and Use
The compositions of the instant invention are useful in medical applications
for treating or preventing a variety of dise~ses including cancer, preferably
metastatic cancer, inflammation, and ~ise~ses caused or exacerbated by platelet
aggregation, or angiogenesis, and for the treatment of conditions or diseases which
are characterized by excessive and destructive smooth muscle cell proliferation.The instant compositions, because of their angiostatic activity, will be
preferably applied for the beneficial treatment of angiogenic-based diseases. One
such class of diseases is cancer or retinopathies. A member of this latter class is
diabetic retinopathy that will be favorably treated by the compositions of the instant
invention.
Administration of the compositions of the invention is typically by routes
appropriate for glycosaminoglycan compositions, and generally includes systemic
administration, such as by injection.
Particularly preferred is subcutaneous injection, as continuous injection over
long time periods can be easily continued. Also preferred are introduction into the
25 vascular system through intraluminal administration or by adventitial administration
using osmotic pumps or implants. Typical implants contain biodegradable materials
such as collagen, polylactate, polylactate/polyglycoside mixtures, and the like.These may be formulated as patches or beads. Typical dosages are in the range
of 0.1 - 100 mg/kg/day on a constant basis over a period of 5-30 days. A
30 particularly preferred dosage is about 0.3 mg/kg/hr, or, for a 70 kg adult, 21 mg/hr
or about 500 mg/day. For certain applications, including cancer, the doses and
WO 9S/09637 2 1 7 1 4 6 5 PCT/US94111368
.
periods of treatment will be chosen by the physician that best fit the need of the
patient.
Other modes of administration are less preferred but maybe more
- convenient. Injection subcutaneously at a lower dose or administered orally at a
slightly higher dose than intravenous injection, or by transmembrane or transdermal
or other topical administration for localized injury may also be effective. Localized
administration through a continuous release device, such as a supporting matrix,perhaps included in a vascular graft material, is particularly useful where the
location of the trauma is accessible.
Formulations suitable for the foregoing modes of administration are known in
the art, and a suitable compendium of formulations is found in Remin~ton's
Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, latest edition.
The compositions of the invention may also be labeled using typical methods
such as radiolabeling, fluorescent labeling, chromophores or enzymes, and used to
15 assay the amount of such compositions in a biological sample following its
administration. Suitable protocols for competitive assays of analytes in biological
samples are well known in the art, and generally involve treatment of the sample, in
admixture with the labeled competitor, with a specific binding partner which is
reactive with the analyte such as, typically, an immunoglobulin or fragment thereof.
20 The antibodies prepared according to the invention, as described below, are useful
for this purpose. The binding of analyte and competitor to the antibody can be
measured by removing the bound complex and assaying either the complex or the
supernatant for the label. The separation can be made more facile by preliminaryconjugation of the specific binding partner to a solid support. Such techniques are
25 well known in the art, and the protocols available for such competitive assays are
too numerous and too well known to be set forth in detail here.
As referred to above, the oligosaccharide compositions of the invention are
useful in therapeutic applications for the treatment of conditions or diseases which
are characterized by excessive and destructive smooth muscle cell proliferation.30 These conditions frequently occur where the subject has been exposed to trauma,
such as in the case of surgical patients. The trauma caused by wounds or surgery
WO 95/09637 PCT/US9`1/11368
~7~465
results in vascular damage and secondary smooth muscle cell proliferation, whichresults in vascular restenosis. This undesirable result can occur after vascular graft
surgery, heart transplantation, balloon or laser angioplasty, arterial traumatic injury,
postsurgical repair of muscular arteries, long-term in-dwelling of arterial catheters,
5 invasive arterial diagnostic procedures, kidney, lung or liver transplants, coronary
artery bypass surgery, carotid artery bypass surgery, femoral popliteal bypass
surgery, and intracranial arterial bypass surgery.
In addition to secondary smooth muscle cell proliferation events occurring as
a result of trauma, certain diseases are associated with unwanted vascular
10 proliferation, although in these cases, too, it is assumed that some internal unknown
injury has caused the secondary result. These disease states include Goodpasturesyndrome, acute glomerulonephritis, neonatal pulmonary hypertension, asthma,
congestive heart failure, adult pulmonary hypertension, and renal vascular
hypertension.
The compositions of the instant invention are useful as inhibitors of smooth
muscle cell proliferation, a preferred application is the inhibition of the proliferation
of smooth muscle cells in blood vessel walls that occurs in response to vascularinjury, and in association with certain dise~se states (Austin, G.E., et al., J Am Coll
Cardiol (1985) 6:369-375). The proliferation of these cells can have negative
20 effects due to the production of excess proteins or other matrix molecules, which,
along with the cells themselves, form pathologic lesions of, for example,
atherosclerosis, renal hypertension, pulmonary hypertension, vasculitis, and
post-surgical vascular restenosis. These results are distinguished from the acute
response to trauma characterized by blood clotting.
In certain of the experiments described herein, the invention compositions
were compared to porcine mucosal heparin. Suitable procedures for the
preparation of the heparin starting material are found, for example, in Charles, A.F.,
et al., Biochem J (1936) 30:1927-1933, and modifications of this basic procedureare also known, such as those disclosed by Coyne, E., in ChemistrY and Biolo~y of
30 Heparin, Elsevier Publishers, North Holland, New York, Lunblad, R.L., et al., eds.
(1981).
WO9S/09637 2 1 7 1 465 PCTIUS94/11368
.
"NAC-antiproliferative heparin" refers to a mixture of non-fragmented
glycosaminoglycan chains obtained by treating commercially available heparin with
periodate as described herein, which mixture substantially lacks anticoagulant
activity but inhibits the proliferation of smooth muscle cells.
Inhibition of Anqioqenesis
A key property of the invention sulfated maltooligosaccharides is that they
exhibit significant anti-angiogenic or angiostatic activity. Angiogenesis is theprocess whereby new blood vessels are produced. It is a process that may be
10 associated with certain dise~.ses, including arthritis, retinopathies, and the growth
and metastasis of tumors. See, Mitchell and Wilks, Annual Reports in Medicinal
Chemistry (Academic Press 1992) 27:139-148; Chapter 15.
Compositions that stimulate or inhibit angiogenesis can be identified using
several assays known in the art. The chick chorioallantoic membrane (CAM) assay
15 is commonly used. Using the CAM assay, certain heparinoids inhibit angiogenesis
when administered with certain steroids. Folkman and Ingber, Ann. Sur~. (1987)
206:374, Folkman et al., Science (1983) 221:719.
The invention compositions described herein also exhibit anti-angiogenic
activity in the CAM assay, and thus can be used to treat angiogenic based
20 dise~ses As mentioned above, one such dise~se is cancer. Thus, while
Applicants do not intend to be held to any one specific mechanism of action to
explain the biological activity of their compositions, the anti-angiogenic activity of the
highly sulfated maltooligos~ccharides would account for, at least in part, their anti-
cancer activity.
Inhibition of Heparanase
The metastatic spread of tumor cells throughout the body is thought to be
facilitated by enzymes secreted by tumor cells that degrade components of the
basement membrane, thereby allowing tumor cells to disseminate via the
30 circulation. One such enzyme is endo-,~-D-glucuronidase, or heparanase, whichdegrades heparan sulfate glycosaminoglycans. Heparan sulfate is a prominent
WO 95/09637 PCT/US94111368
~7~b5 ` ~
component of parenchymal cell basement membranes. The highly sulfated
maltooligosaccharides of the instant invention exhibits significant heparanase
inhibitory activity as revealed using standard assays. Thus, cancer, and other
diseases that have as one element unwanted heparanase activity can be
5 beneficially treated with the compositions of the invention.
Inhibition of bFGF ActivitY
Basic fibroblast growth factor (bFGF) is a small, heparin-binding polypeptide
growth factor which is mitogenic for a variety of cell types of meso- and
10 neuroectodermal origin. The mitogenic activity of bFGF is believed to derive from
its specific interaction with one or more high affinity transmembrane receptors in the
tyrosine kinase gene family.
Basic FGF is also known to interact with cell surface and extracellular matrix
heparan sulfate proteoglycan (HSPG), and such molecules are often referred to as15 "low affinity" receptors. The protein also binds heparin quite strongly in vitro, and
advantage of this is taken in the routine purification of bFGF on affinity columns of
immobilized heparin. More recently, several studies have provided evidence that
heparin or the heparan sulfate (HS) chains of HSPG may in fact be a cofactor that
promotes or enhances the binding of bFGF to its high affinity receptors.
The bFGF binding properties of certain heparins or heparin like molecules
are described by Ishihara, M., et al., Anal Biochem (1992) 202:310-315.
The capacity of the invention compositions to bind to bFGF and inhibit bFGF
dependent cell growth can be shown using certain assays. Assays for measuring
the effect of heparinoids on bFGF are known in the art. A cell based competitivebinding assay is described by Ishihara, M., et al., Anal Biochem (1992) 202:310-315. The assay is based on the observation that bFGF binds to a Iymphoblastoid
cell line, transfected with hamster syndecan (RO-12 UC cells), and that this
interaction can be inhibited by compositions that bind to bFGF.
A key aspect of the invention compositions is that the inhibition of bFGF-
30 dependent cell growth is dependent on the degree of sulfation of the
maltooligosaccharide compositions, the highly sulfated compositions described
W095/09637 2 1 7 1 465 PCT/US94/11368
.
herein being the most effective.
Platelet Inhibition
Heparin is known to have an anti-thrombotic effect, and at least in part this is
5 a result of heparin's capacity to inhibit platelet aggregation. Interference with
platelet aggregation causes a significant bleeding liability in some patients. Certain
NAC heparins exhibit both non-anticoagulant activity and inhibit platelet
aggregation. See, for example, co-owned U.S. Patent Application, Serial No.
753,299, filed September 3, 1991, or PCT Patent Application No. US92/02516, filed
10 March 27, 1992. The preferred compositions similar to heparin, of the instant invention inhibit platelet aggregation.
The following examples are intended to illustrate but not to limit the invention.
For example, those skilled in the art would know that there are materials and
methods that can be substituted for those described below, and still come within the
15 scope of what is detailed in the Examples.
EXAMPLES
Example 1
Effect of Sulfation of Maltooliqosaccharides on Cell Bindinq and Proliferation
To study the effect of the degree of sulfation on the biological properties of
maltooligos~ccharides, maltohexaose was sulfated with 6, 12, and 18 moles per
mole of maltohexaose with sulfur trioxide pyridine complex to give Compositions 1,
2, and 3 in Table 1, respectively. Composition 4, the most highly sulfated
composition, was generated using an excess (2 moles/hydroxyl group) of the sulfur
25 trioxide pyridine complex in DMF.
The average degree of sulfation of the products was deduced from the data
of elementary analysis. Because of the hygroscopic nature of the products the
carbon/sulfur ratio was used instead of the sulfur content.
More specifically, sulfation was carried out as follows: Compositions 1, 2
30 and 3 were made in three parallel experiments. To a solution of maltohexaose
(0.198 g) in N,N-dimethylformamide (10 mL) sulfur trioxide pyridine complex (0.191
WO 95/09637 PCT/US94/11368
465 ' ~
g (Experiment 1), 0.382 9 (Experiment 2), and 0.5739 (Experiment 3), respectively)
was added. The mixtures were stirred at room temperature for two days. The pH
was adjusted to 9 with 1M NaOH, and the mixtures were evaporated to dryness
under reduced pressure. The crude products were desalted on a Biogel P-2 column
5 with 0.5M NH4HCO3 as eluents, then were further purified by passing through a
column of SP Sephadex C25 (Na') ion-exchange gel with water as eluent to give
0.110g (Experiment 1), 0.1879 (Experiment 2), and 0.3509 (Experiment 3) product,respectively. Compositions 1, 2 and 3 were produced in Experiments 1, 2 and 3,
respectively.
Composition 4, highly sulfated maltohexaose was made as follows:
Maltohexaose (0.59) in N,N-dimethylformamide (20 mL) was treated with sulfur
trioxide pyridine complex (3.21 g) and the mixture was stirred at room temperature
for 2 days. The pH was adjusted to 9 with 1 M NaOH, and the product was
precipitated by the addition of ethanol. The crude product was filtered and was
desalted on a Biogel P-2 column with 0.5M NH4HCO3, then passed through a
column of AG 50W-X8 (Na~) ion-exchange resin with water, to give 1.249 material
after Iyophilization. The results are summarized in Table 1.
Table 1
Deqree of Substitution of Sulfated Maltohexaose
CG~ JO~jI;On Deqree of # OH GrouPs
C/S Ratio Sulfation in Parent C~l,.".,~-~tion
Found Calc~ t~J
6.12 6.74 2 2
2 2.37 2.25 6 20
3 1.20 1.22 11 20
4 0.90 0.90 15 20
The effect of Compositions 1, 2, 3 and 4 on the binding of RO-12 UC cells to
bFGF coated micro-titer wells was determined as described by Ishihara, M., et al.,
Anal Biochem (1992) 202:310-31~. Bound cells are readily quantitated as total
35 protein. Heparin which inhibits RO-12 UC cell binding was run as a positive control.
14
W095/09637 ~ ~ 7 1 ~6~ PCT/US94111368
.
The assay was run as follows: Fifty microliters of 10 ug/ml human
recombinant bFGF was added to wells of a 96-well tissue culture plate and
incubated overnight at 4C. The wells were aspirated with PBS to remove any
unbound bFGF, rinsed twice with PBS, and subsequently incubated with PBS
5 containing 5% (v/v) fetal bovine serum for 1 hour at room temperature. RO-12 UC
cells were suspended at a density of 3 x 106 cells/ml in PBS containing 5% fetalbovine serum. To this mixture was added the desired amount of sulfated
composition, or heparin. They were made up in PBS plus 2.5% fetal bovine serum.
A control was also run, containing only PBS plus 2.5% fetal bovine serum. Next,
10 100 ul of the cell suspension was immediately added to the microtiter wells, and
incubated for 5 minutes, after which the wells were washed 3 times with PBS.
Finally, the amount of cell protein bound to the wells was determined by dissolving
the bound cells in 20 ul of 5% SDS and measuring the protein concentration of the
cell Iysates. BSA was used as the standard.
15The results are shown in Figure 1. It is apparent that the more highly
sulfated the maltohexaose is, the greater its ability to block RO-12 UC cell adhesion
to bFGF-coated wells.
To extend the effects seen with RO-12 UC cells, a second experiment was
conducted. The capacity of the sulfated maltohexaoses to inhibit the proliferation of
20 a bFGF-dependent adrenocortical endothelial (ACE) cell line was determined. This
cell line (provided by D. Gospodarowicz, UCSF) requires either aFGF or bFGF for a
proliferative response. Cells were seeded at low density in microtiter wells in the
presence of 2 ng/ml bFGF, and growth was determined as total protein after four
days in the presence of the sulfated maltohexaoses. The results are shown in
25 Figure 2. Again, the more highly sulfated maltohexaose compositions exhibited the
highest anti-proliferative activities.
Example 2
SYnthesis of Sulfated Maltotetraose and Bioloqical Activity
30Maltotetraose may be purchased commercially from Sigma Corporation or
can be produced enzymatically using the procedure of Ratanakhanockchal et al.
WO 9S/09637 PCT/US94/11368
?~7~465 ~
See, Applied and Environmental Microbiology: vol. 58, no. 8, pages 2490-2494.
Maltotetraose is sulfated as follows.
Maltotetraose (0.50 9) in N,N-dimethylformamide (20 mL) was treated with
sulfur trioxide pyridine complex (3.34 9) at room temperature. A brownish syrup
5 precipitated in about 15 minutes. The mixture was stirred for 2 days at room
temperature then was cooled to 0C. The pH was adjusted to approximately 9 with
1 N NaOH. The product was precipitated with ethanol, and the solid was filtered
and washed with ethanol. The crude product (3.22 g) was desalted on a Biogel P-2column with 0.5M NH4HCO3. Lyophilization of the carbohydrate containing fractions
10 gave 1.44 9 off-white solid. This material was passed through a column of AG
W50-X8 (Na+) ion-exchange resin using water as eluent to give 1.51 9 product.
The degree of sulfation of the product, denoted Composition 5, is shown in Table 2.
Table 2
Deqree of Substitution of Malll~lelraose
Co"".~itionDeqree of # OH GrouPs
CIS Ratio Sulfation in rar~ CG."Po~ilion
Found Calu l~te~
0.69 0.69 13 14
The capacity of sulfated maltotetraose to inhibit cell binding and cell
proliferation was described using the assays set forth in Example 1 and the results
are shown in Figures 3 and 4. It is apparent that this composition has significant
activity in both assays.
Example 3
SYnthesis of Hiqhlv Sulfated Maltopentaose and Maltoheptaose
Maltopentaose and maltoheptaose were obtained from Sigma Corporation,
and sulfated using essentially the reaction conditions described for the sulfation of
maltotetraose in Example 2. Maltoheptaose can also be prepared by the acetolysis
WO 95/09637 2 1 7 1 4 6 5 PCT/US94/11368
.
of ,~-cyclodextrin as described by N. Sakairi et al., J. Chem. Soc. Chem. Commun.,
(1991) 289. The degree of sulfation of both compositions, maltopentaose and
maltoheptaose, is shown in Table 3 as Compositions 6 and 7, respectively.
Table 3
Deqree of Substitution of Sulfated Malto~ ao~/MaltohePtaose
and Bioloqical Activities
C~ ition Deqree of # OH Groups
C/S Ratio Sulfation in Parent Cu~ J&-ition
Found Calculated
6 1.29 1.25 9 17
7 0.91 0.93 17 23
The capacity of highly sulfated maltopentaose and highly sulfated
maltoheptaose to inhibit cell binding and cell proliferation was tested using the
20 assays se~ forth in Example 1, and the results are shown in Figures 3 and 4,
respectively. It is apparent that both compositions, similar to highly sulfated
maltotetraose, show significant activity in both assays.
Example 4
SYnthesis of Hiqhly Sulfated Maltose
and Maltotriose and Bioloqical Activities
To determine if the size of highly sulfated maltooligosaccharides is an
important factor in regulating cell growth or cell binding, maltose and maltotriose
were sulfated and tested in the assays described above.
Maltotriose may be purchased commercially or can be produced
enzymatically using the procedure of Ratanakhanockchal et al. See Applied and
Environmental Microbiology:58, no; 8, pages 2490-2494. Maltotriose was sulfated
as follows. Maltotriose and maltose were sulfated using essentially the reactionconditions described for maltotetraose. In the case of maltose, after adjusting the
35 pH to 9 the product was not collected by precipitation with ethanol, but by
WO 9S/09637 PCT/US94/11368
465
concentrating the reaction mixture under reduced pressure. The degree of
substitution of the Composition 8 is shown in Table 4.
Table 4
Deqree of Substitution of Maltotriose
ComPositionDeqree of # OH Groups
C/S Ratio Sulfation in Parent con-Positlon
Found Calculated
8 0.69 0.67 10 11
The capacity of sulfated maltotriose to inhibit cell binding and cell
proliferation was tested using the assays set forth in Example 1 and the results are
shown in Figures 3 and 4, respectively. It is apparent that this composition shows
little or no activity in both assays. Maltose was sulfated to a high degree and
tested in the cell binding and cell proliferation assays described in Example 1. The
20 degree of sulfation is shown in Table 5, and the biological data are presented in
Figures 3 and 4, respectively. Note that this composition, denoted Composition 9 in
the table, has little or no activity relative to the highly sulfated maltotetraose
composition, and the other larger highly sulfated oligosaccharides shown in the
figures.
Table 5
DeQree of Substitution of Mdllose
Cc~ r ~ition De~ree of # OH GrouPs
C/S Ratio Sulfation in Parent Con".osilion
Found Calculated
9 0.64 0.64 7 8
Based on these results shown here and in Examples 1-3, it is apparent that
both the size and degree of sulfation of maltooligosaccharides affect cell binding
18
W095/09637 21 7 1 ~5 PCT/US94/11368
.
and cell proliferation. Highly sulfated maltooligosaccharides where n24 are the
most effective in both assays. In contrast, highly sulfated maltotriose and highly
sulfated maltose are less active in both assays.
Example 5
Effects of Hiqhly Sulfated Maltotetraose on Tumor Growth
Experiments were conducted to test the efficacy of highly sulfated
maltotetraose, Composition 5, Table 2, on tumor growth in an animal model system,
the nude mouse. The growth of human tumors in the nude mouse has clinical
10 relevance to the growth of tumors in humans.
Three human tumor cell lines were utilized, the mammary adenocarcinoma.
MDA231, the pancreatic tumor cell line, CAPAN-2, and the prostatic
adenocarcinoma cell line, PC-3. All cell lines are available from the American Type
Culture Collection, and grow aggressively in nude mice. The experiments were
15 conducted as follows: female, 15-20 gram nude mice, in groups of ten, were
inoculated subcutaneously with 3-5 x 106 viable MDA231, CAPAN-2, or PC-3 cells
in 0.1 ml PBS. The cells were grown in standard DMEM tissue culture media
supplemented with 10% fetal calf serum in a humidified 5% C02 incubator at 37C.The cells were harvested with trypsin-EDTA, washed 2x with PBS, resuspended at
20 a concentration of 3-5 x 107 cells/ml, and placed on ice prior to injection.
On a daily basis, the mice were subcutaneously injected with 100 mg/kg of
highly sulfated maltotetraose made up in PBS, and produced as described in
Example 2. The composition was filter sterilized with a 0.2um Gelman filter unit.
Tumor volume was determined using the following formula:
Tumor Volume = Lenqth x Width2
The effects of sulfated maltotetraose on MDA231 tumor growth are shown in
30 Figure 5. Control mice were injected with PBS vehicle only. Experimental and
control mice were injected twice daily with 0.05 ml of the appropriate solution
starting on days 0-40 post tumor challenge. From days 41-70 the animals were
19
WO 95/09637 2. ~ 7 ~ ~ 6~ PCT/US9~/11368
dosed once daiiy with 100mg/kg/day in a volume of 0.1 ml per injection, and tumor
volume measured at defined times using standard methods.
Referring to Figure 5, it is apparent that tumor growth is significantly
inhibited. Inhibition is first apparent at about days 36-37, and is dramatic by days
5 68-69. Note that from about day 59 until the end of the experiment (day 70),
inhibition was significant at p values of <0.05. The tumor volume at days 68-69 in
control and sulfated maltotetraose treated animals is about 37smm3 and 125mm3,
respectively.
The effects of highly sulfated maltotetraose on CAPAN-2 tumor cell growth
10 were determined using the materials and methods set forth above with the following
exceptions. Two groups of 20 female nude mice were used for the experimental
and control groups, and the animals were dosed once daily starting on day 0 of
tumor challenge. The results are shown in Figure 6. It is apparent that this
composition also significantly inhibits tumor growth. Inhibition is observed at days
15 20-34 and 41-48. The experiment was run for 52 days.
Finally, the effects of highly sulfated maltotetraose on the human prostatic
adenocarcinoma line, PC-3, was determined. This cell line is also available fromthe American Type Culture Collection. The materials and methods described above
were similarly used here with the following exceptions. The composition was
20 administered subcutaneously in a single dose of 80 mglkgl day in a volume of 0.05
ml per dose Animals were dosed beginning 24 hours after tumor challenge. 5 x
106 PC-3 cells were injected in a volume of 0.1 ml subcutaneously in the anterior
dorsal region (12 animals/treatment group).
The experiment was conducted for 23 days. The results are shown in Figure
25 7. The highly sulfated maltotetraose composition inhibited PC-3 cell growth at all
the time points tested. Note that at days 15, 19, and 21 growth inhibition was
significant at p values of p=0.06, 0.058, and 0.093, respectively.
WO 95/09637 2 ~ 5 PCT/US94/11368
.
ExamPle 6
Inhibition of Smooth Muscle Cell Growth
Maltotetraose, maltopentaose and maltohexaose were sulfated as described
in Examples 1-3, and assayed for their effects on smooth muscle cell proliferation
5 using a standard assay for this activity. A convenient assay, in detail, is as follows:
Solutions to be tested are made up in DMEM medium containing 10% fetal
calf serurn and penicillin/streptomycin. Bovine smooth muscle cells (SMC) are
isolated from bovine aorta by the method of Ross, R., J Cell Biol (1971) 172-186.
SMC from passage 3-10 are plated at 350-700 cells per well in 96-well microtiter10 plates in the medium above and allowed to attach for 2-4 hr. The complete
medium is then replaced with DMEM supplemented with 0.1% fetal calf serum, and
the cells are incubated for an additional period of about 24 to 72 hr to arrest cell
growth. The low-serum medium is then replaced with complete medium containing
the test samples. The sulfated maltose oligosaccharides of the invention were
15 added to the medium to make final concentrations as shown in the figures. Theeffects of heparin at the same concentrations were also determined. The results
are plotted as the per cent of heparin inhibition where the concentration of heparin
that gave 100% inhibition of SMC proliferation was 150 ug/ml.
The cells are allowed to grow for up to 7 days with replicate plates sampled
20 at regular intervals. Cell number is determined by removing the medium and
washing the cells with phosphate-buffered saline, adding 75-150 ul lysis buffer, and
assaying for lactate dehydrogenase (LDH) activity, as described by Brandley, B., et
al., J Biol Chem (1987) 262:6431. The activity of LDH is proportional to cell
number.
The results are shown in Figures 8-10. Figure 8 shows the anti-proliferative
activity of highly sulfated maltotetraose. This composition exhibits significant anti-
proliferative effects, although not as great as heparin. With respect to heparin,
about 50% inhibition is observed at about 60 ug/ml.
Figure 9 shows the anti-proliferative activity of highly sulfated maltopentaose.30 This composition is more effective in inhibiting SMC proliferation than the smaller-
sized sulfated maltotetraose. Indeed, 100% inhibition of SMC growth is observed at
21
WO 95/09637 PCT/US9~/11368
465 ~
about 50 ug/ml. In contrast, one hundred per cent inhibition was not observed for
highly sulfated maltotetraose at a concentration of 150 ug/ml.
Figure 10 shows the anti-proliferative activity of highly sulfated maltohexaose.This composition shows similar efficacy in comparison to highly sulfated
5 maltopentaose. These date show that highly sulfated maltooligosaccharides of the
invention are inhibitors of SMC growth, and thus, can be beneficially applied for the
treatment of diseases where SMC growth is sought to be controlled or eliminated.
Example 7
Anqiostatic Activity
Compositions that stimulate or inhibit angiogenesis can be identified using
several assays known in the art. Highly sulfated maltotetraose, Composition 5,
Table 2, was tested for angiogenesis activity using the chicken chorioallantoic
membrane (CAM) assay. The assay was performed as described by Castellot et.
15 al., J. of Cellular Phvsioloqy (1986) 127: 323-329, with the exception that samples
were evaluated for their efficacy to inhibit neovascularization. Carboxy methyl
cellulose pellets containing 50 ug of hydrocortisone, or hydrocortisone plus different
amounts of highly sulfated maltotetraose were incubated on the CAM for 3-4 days
before scoring the results. Highly sulfated maltotetraose was produced as
20 described in Example 2. Angiostatic activity is defined as a partial clearing or an
avascular zone around the pellet. In all cases, pellets at each maltooligosaccharide
concentration contained 50 ug of hydrocortisone. The number in the Table is the
number of embryos scored that exhibited no effect, a partial clearing (+), or anavascular zone (++). Table 6 shows the results.
It is apparent that the sulfated composition exhibits angiostatic activity. At
low concentrations (eg. 3 and 6 ug/mL), the composition is slightly better at
inhibiting the formation of new blood vessels than the buffer control, or
hydrocortisone alone. At the higher concentrations, 12.5, 25, and 50 ug/ml,
increase in partial clearing areas and avascular zone are observed.
W095/09637 2 1 7 ~ 46~ PcTrusg4lll368
.
Table 6
Anqiostatic Activity of Hiqhly Sulfated Mall~l~lrdose (HSM)
NO
SAMPLE EFFECT ~ ++
Buffer Control 39 0 0
I Iy.l~ o~ortisone 27 2 0
50 ug/pellet
CG.",.osilion 4 t Hydrocortisone
50 ug/pellet 6 2 4
9 11 2
12.5 11 10 3
6 16 4 0
3 11 1 0
Scoring done at 24hr
Pellets = 0.5% 3, leous ~lh~ lose
Buffer control - ddH20
HSM alone was inactive
Example 8
Inhibition of Heparanase
Highly sulfated maltotetraose, Composition 5, Table 2, maltoheptaose and
maltose were tested for heparanase inhibitory activity using heparanase from a rat
hepatoma cell line. The cell line is described by Gerschenson, et al., Science
(1970) 170: 859-861. Further, their inhibitory activities were compared to porcine
40 mucosal heparin.
The procedures for isolating heparanase from hepatoma cells, and the methods forassaying the activity of the enzyme are known by those skilled in the art. The
following procedures and materials were used. Confluent rat hepatoma cell cultures
23
WO 9S/09637 PCT/US94/11368
465 ~
were grown in standard cell culture flasks, and washed 3 times with 10 ml of a 50
mM Hepes solution containing 0.25M sucrose and 0.14 M NaCI, pH 7.4. Next, 1ml
of a 50 mM MES buffer, pH 5.2, containing 0.14M NaCI, 6 mM sodium azide, and
certain protease inhibitors was added and the cells removed from the flask using a
5 disposable cell scraper. The following protease inhibitors were present in the MES
buffer: 0.2 ug/ml aprotinin, 0.5 ug/ml leupeptin,100 ug/ml soybean trypsin inhibitor,1
mM PMSF, 2 mM EDTA (sodium salt), and 15mM D-saccharic acid 1,4 lactone
(exoglucuronidase inhibitor).
The cells were added to a 7ml Dounce homogenizer, freezed/thawed 3 times
10 in an ethanol/dry ice bath, and homogenized with 15 strokes using a tight pestle.
The resulting cell Iysates were placed in a 2 ml centrifuge tube and centrifuged at
4 C for 30 minutes at 16,000 x 9. The supernatant was removed, and the protein
concentration in the supernatant determined using the Macro BCA protein assay.
BSA was used as a standard. Heparanase activity was quantified by measuring
15 soluble N-3H-acetylated pancreas heparan sulfate fragments derived from
uncleaved N-3H-acetylated pancreas heparan sulfate distinguishable by
cetylpyridinium chloride (CPC) precipitation. N-3H-acetylated pancreas heparan
sulfate had a weight average molecular weight, or Mw, of about 13,000. The
following procedures were used.
3H-acetylated pancreas heparan sulfate (248 ng, 80,000 cpm) in 10 ul of 200
mM MES buffer, pH 5.2, containing 0.14M NaCI was added to 1.5 mL siliconized
microcentrifuge tubes. Next, 10 ul of distilled water containing various
concentrations of porcine mucosal heparin, or the appropriate highly sulfated
maltooligosaccharide, was added. Then, 30 ul of rat hepatoma cell supernatant,
isolated as described above, containing 7.5 - 10 ug of protein in 50mM MES buffer,
pH 5.2, containing 0.14M NaCI, 6mM sodium azide and the protease inhibitors
described above, is added to siliconized 1.5 ml microcentrifuge tubes. Three to six
replicates were run for each concentration of highly sulfated maltotetraose while
three replicates were run for each concentration of porcine mucosal heparin.
30 Controls were run to account for background counts. It was previously shown that
the highest concentration of inhibitor does not affect precipitation of the intact
24
W095/09637 2 1 7 1 ~65 PCT/US94111368
.
radiolabeled heparan sulfate substrate.
The enzyme substrate inhibitor mixture was mixed, after which the tubes
were incubated at 37C for 20 minutes. After 20 minutes, the reaction was stopped
by adding to the reaction tubes 150 ul of an aqueous heparin solution (0.33 mg/ml).
5 200 ul of 100mM sodium acetate pH 5.5 and 100 ul of CPC t0.6% in water) were
then added. Three replicates maintained on ice were run as background controls (0
min.) in which the enzyme was added to the tubes immediately followed by heparinto terminate the reaction. The samples were processed as described for the 20
minute time points. Next, the tubes were vortexed, incubated for 60 minutes at
10 room temperature, and then centrifuged for 10 minutes at 4,000 x g in a 5415CEppendorf centrifuge. The supernatant was removed and assayed for 3H by liquid
scintillation counting.
Values represented are the mean differences between soluble CPM
measured at 20 minutes at different heparin or highly sulfated maltotetraose,
15 maltoheptaose, and maltose concentrations and soluble CPM measured at 0
minutes at 0 ug/ml +/- the standard deviation of the sum of the 20 and 0 minute
variances.
Figure 11 shows that highly sulfated maltotetraose is approximately one half
as effective as porcine mucosal heparin in inhibiting heparanase activity. The IC50
20 values of highly sulfated maltotetraose and porcine mucosal heparin were 9.0 and
4.1 ,ug/ml, respectively.
Not shown in the figure are the results for maltoheptaose sulfate and maltose
sulfate. The former was as active as heparin in the assay, whereas highly sulfated
maltose had no activity at greater than 200 ug/ml.
Example 9
SYnerqistic Effect of Hiqhly Sulfated Maltooliqosaccharides
and ChemotheraPeutic Druqs in the Treatment of Cancer
As discussed above, one mechanism whereby the highly sulfated
30 maltooligosaccharides exert their anticancer effect is by blocking angiogenesis.
Thus, experiments would be done to show a synergistic effect of these
wo 95/o~6~77 ~ 4 65 PCT/US9~/11368
compositions with chemotherapeutic drugs that are known to be effective for
treating cancer.
Such experiments would be done as described in Example 5 using the tumor
cell line MDA231, but having an additional test sample consisting of methotrexate.
5 The procedure would be as follows. On a daily basis, mice would be
subcutaneously injected with 100 mg/kg of highly sulfated maltotetraose,
Composition 5, Table 2, made up in PBS, produced as described in Example 2,
with methotrexate. Methotrexate would be used at a concentration of 4 mg/kg.
The composition would be filter sterilized with a 0.2um Gelman filter unit.
Similar to Example 5 tumor volume would be determined using the following
formula:
Tumor Volume = Lenqth x Width2
Three groups of control mice would be run. They would be injected with
either PBS vehicle, 100 mg/kg of highly sulfated maltotetraose in PBS, or
4 mg/kg methotrexate in PBS. Experimental (highly sulfated maltotetraose with
methotrexate) and control mice would be injected twice daily with 0.05 ml of the20 appropriate solution starting on days 0-40 post tumor challenge. From days 41-70
the animals would be dosed once daily with 100mg/kg/day in a volume of 0.1 ml
per injection, and tumor volume measured at defined times using standard
methods.
The effects of highly sulfated maltotetraose alone on MDA231 tumor growth
25 would be as shown in Figure 5. That is, tumor growth would be significantly
inhibited with inhibition first apparent at about days 36-37, and dramatic inhibition
apparent by days 68-69. The tumor volume at days 68-69 in control (PBS) and
highly sulfated maltotetraose treated animals would be about 375mm3 and 125mm3,
respectively.
In the second group of control mice treated with methotrexate alone, similar
results would be expected as those observed for mice treated with highly sulfated
maltotetraose alone. That is, there would be a significant reduction in tumor volume
2~714G5
WO 9~/09637 PCT/US94/11368
.
relative to the PBS control mice; about a 3 fold reduction would be realized.
In contrast, however, mice treated with the combination of highly sulfated
maltotetraose and methotrexate would present an average tumor volume less than
that observed with either agent alone. Tumor volume would be less than 60mm3 at
5 about days 68-69.
Having described what the applicants believe their invention to be, a skilled
practitioner of this art should not construe the invention to be limited other than by
the scope of the appended claims.
