Angiogenesis inhibitors and use thereof

The present invention is based on the unexpected discovery that a molecule having as its major repeating units N-acetylglucosamine alternating in sequence with 2-O-sulfated uronic acid, inhibits FGF mitogenicity, and thus is useful in inhibiting angiogenesis. Additionally, the molecule has low toxicity and inhibits FGF mitogenicity without affecting anticoagulant activity. One preferred molecule is a glycosaminoglycan such as archaran sulfate. The molecules are in pharmaceutical compositions that can be used in the treatment of diseases which are angiogenesis-dependent.

BACKGROUND OF THE INVENTION 
Blood vessels are the means by which oxygen and nutrients are supplied to 
living tissues and waste products are removed from living tissue. 
Angiogenesis refers to the process by which new blood vessels are formed. 
See, for example, the review by Folkman and Shing, J. Biol. Chem. 267 
(16), 10931-10934 (1992). Thus, where appropriate, angiogenesis is a 
critical biological process. It is essential in reproduction, development 
and wound repair. However, inappropriate angiogenesis can have severe 
negative consequences. For example, it is only after many solid tumors are 
vascularized as a result of angiogenesis that the tumors have a sufficient 
supply of oxygen and nutrients that permit it to grow rapidly and 
metastasize. Because maintaining the rate of angiogenesis in its proper 
equilibrium is so critical to a range of functions, it must be carefully 
regulated in order to maintain health. The angiogenesis process is 
believed to begin with the degradation of the basement membrane by 
proteases secreted from endothelial cells (EC) activated by mitogens such 
as vascular endothelial growth factor (VEGF) and basic fibroblast growth 
factor (bFGF). The cells migrate and proliferate, leading to the formation 
of solid endothelial cell sprouts into the stromal space, then, vascular 
loops are formed and capillary tubes develop with formation of tight 
junctions and deposition of new basement membrane. 
In adults, the proliferation rate of endothelial cells is typically low 
compared to other cell types in the body. The turnover time of these cells 
can exceed one thousand days. Physiological exceptions in which 
angiogenesis results in rapid proliferation typically occurs under tight 
regulation, such as found in the female reproduction system and during 
wound healing. 
The rate of angiogenesis involves a change in the local equilibrium between 
positive and negative regulators of the growth of microvessels. The 
therapeutic implications of angiogenic growth factors were first described 
by Folkman and colleagues over two decades ago (Folkman, N. Engl. J. Med., 
285:1182-1186 (1971)). Abnormal angiogenesis occurs when the body loses at 
least some control of angiogenesis, resulting in either excessive or 
insufficient blood vessel growth. For instance, conditions such as ulcers, 
strokes, and heart attacks may result from the absence of angiogenesis 
normally required for natural healing. In contrast, excessive blood vessel 
proliferation can result in tumor growth, tumor spread, blindness, 
psoriasis and rheumatoid arthritis. 
There are instances where a greater degree of angiogenesis is 
desirable--increasing blood circulation, wound healing, and ulcer healing. 
For example, recent investigations have established the feasibility of 
using recombinant angiogenic growth factors, such as fibroblast growth 
factor (FGF) family (Yanagisawa-Miwa, et al., Science, 257:1401-1403 
(1992) and Baffour, et al., J Vasc Surg, 16:181-91 (1992)), endothelial 
cell growth factor (ECGF) (Pu, et al., J Surg Res, 54:575-83 (1993)), and 
more recently, vascular endothelial growth factor (VEGF) to expedite 
and/or augment collateral artery development in animal models of 
myocardial and hindlimb ischemia (Takeshita, et al., Circulation, 
90:228-234 (1994) and Takeshita, et al., J Clin Invest, 93:662-70 (1994)). 
Conversely, there are instances, where inhibition of angiogenesis is 
desirable. For example, many diseases are driven by persistent unregulated 
angiogenesis, also sometimes referred to as "neovascularization." In 
arthritis, new capillary blood vessels invade the joint and destroy 
cartilage. In diabetes, new capillaries invade the vitreous, bleed, and 
cause blindness. Ocular neovascularization is the most common cause of 
blindness. Tumor growth and metastasis are angiogenesis-dependent. A tumor 
must continuously stimulate the growth of new capillary blood vessels for 
the tumor itself to grow. 
The current approved treatment of these diseases is inadequate. Agents 
which prevent continued angiogenesis, e.g, drugs (TNP-470), monoclonal 
antibodies, antisense nucleic acids and proteins (angiostatin and 
endostatin) are currently being tested, but have not been approved. See, 
Battegay, J. Mol. Med., 73, 333-346 (1995); Hanahan et al., Cell, 86, 
353-364 (1996); Folkman, N. Engl. J. Med., 333, 1757-1763 (1995). Although 
preliminary results with the antiangiogenic proteins are promising, they 
are relatively large in size and are difficult to use and produce. 
Moreover, proteins are subject to enzymatic degradation. Thus, new agents 
that inhibit angiogenesis are needed. New antiangeogenic proteins or 
peptides that show improvement in size, ease of production, stability 
and/or potency would be desirable. 
Members of the FGF family are characterized by their high affinity for 
glycosaminoglycan and heparin, and their high mitogenicity for mesodermand 
neuroectoderm derived cells. Furthermore, they are among the most potent 
inducers of neovascularization (see Kan, M. et al., "An Essential 
Heparin-binding Domain in the Fibroblast Growth Factor Receptor Kinase", 
Science, Volume 259, (Mar. 26, 1993) pp. 1918-1921; Ornitz, D. M. et al. 
"Heparin is Required for Cell-free Binding of basic Fibroblast Growth 
Factor to a Soluble Receptor and for Mitogenesis in Whole Cells", 
Molecular and Cellular Biology, Volume 12, (January 1992) pp. 240-247; 
Klagsbrun, M. et al. "MINIREVIEW: A Dual Receptor System is Required for 
Basic Fibroblast Growth Factor Activity", Cell, pp. 229-231; Risau, W., 
"Angiogenic Growth Factors", Progress in Growth Factor Research, Volume 2, 
(1990) pp. 71-79; Bouck, N., "Tumor Angiogenesis: The Role of Oncogenes 
and Tumor Suppressor Genes", Cancer Cells, Volume 2, Number 6, (June 1990) 
pp. 179-185). 
Several inhibitors of FGF-2 mitogenic activity have been described, 
including the synthetic polymers sulfated beta-cyclodextrins, sulfated 
malto-oligosaccharides and phophororothioate oligodeoxynucleotides as well 
as the drug suramin (Guimond, et al., Biol. Chem. 268, 23906-23914, 
Venkataraman, et al., Proc. Natl. Acad. Sci., USA 93, 845-850). While 
these inhibitors have been proposed to be anti-angiogenic agents, 
potentially useful in cancer chemotherapy and in preventing restenosis 
following vascular injury, their use as FGF-2 antagonists has been limited 
because of their anticoagulant potency or in vivo toxicity. 
Thus, there is a need for FGF antagonists having low toxicity and 
anticoagulant activity. 
SUMMARY OF THE INVENTION 
The present invention is based on the unexpected discovery that a molecule 
having as its major repeating units N-acetylglucosamine alternating in 
sequence with 2-O-sulfated uronic acid, inhibits FGF mitogenicity, and 
thus is useful in inhibiting angiogenesis. Additionally, the molecule has 
low toxicity and inhibits FGF mitogenicity without affecting anticoagulant 
activity. One preferred molecule is a glycosaminoglycan such as archaran 
sulfate. The molecules are in pharmaceutical compositions that can be used 
in the treatment of diseases which are angiogenesis-dependent. 
Angiogenesis-dependent diseases include, but are not limited to retinal 
neovascularization, tumor growth, hemogioma, solid tumors, leukemia, 
metastasis, psoriasis, neovascular glaucoma, diabetic retinopathy, 
rheumatoid arthritis, endometriosis, and retinopathy of prematurity (ROP). 
The present invention relates to a method of inhibiting angiogenesis in a 
host in need thereof comprising administering to the host an angiogenesis 
inhibitory effective amount of, for example, a glycosaminoglycan having as 
its major repeating unit N-acetylglucosamine alternating in sequence with 
2-O-sulfated uronic acid. 
Other aspects of the invention are disclosed infra.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention includes compositions and methods for the treatment 
of diseases that are mediated by angiogenesis. One embodiment of the 
present invention is the use of a molecule having as its major repeating 
unit N-acetylglucosamine alternating in sequence with 2-O-sulfated uronic 
acid and having FGF antagonist activity to inhibit unwanted angiogenesis. 
Preferably the molecule is a glycosaminoglycan. The present invention 
comprises a method of treating undesired angiogenesis in a human or animal 
comprising the steps of the administering to the human or animal with the 
undesired angiogenesis a composition comprising an effective amount of, 
for example, a glycosaminoglycan having as its major repeating unit 
N-acetylglucosamine alternating in sequence with 2-O-sulfated uronic acid. 
Preferably, the glycosaminoglycan is at least about 6 monomers, more 
preferably at least about 10 monomers, still more preferably at least 
about 12 monomers. The number of monomers preferably ranges from 12-75 
monomers. More preferably, the glycosaminoglycan is 12-50 monomers. Most 
preferably, the glycosaminoglycan is 12-35 monomers. Such 
glycosaminoglycans can be obtained using known methods in the art. 
Preferred glycosaminoglycans include archaran sulfate and heparin lyase 
I-resistant regions in the heparan sulfate chains of syndecan-1 (Kato et 
al., Nature Medicine 4,691-697 (1998)). 
Acharan sulfate is a novel glycosaminoglycan of the structure, composed 
primarily of the repeating unit, .fwdarw.4)GlcNpAc(1.fwdarw.4) 
IdoAp2S(1.fwdarw.where Ac is acetate) (FIG. 1). Acharan sulfate was 
isolated from the giant African snail Achatina fulica and can be obtained 
by the method set forth in Kim et al., J. Biol. Chem. 271, 1116-1120 
(1996), the disclosure of which is herein incorporated by reference. Low 
molecular weight archaran sulfate can be prepared using, for example, the 
method set forth in Example 2. Chemical modification of this 
glycosaminoglycan converted it into a polysaccharide (N-sulfoacharan 
sulfate) with the predominant structure 
.fwdarw.4)GlcNpS(1.fwdarw.4)IdoAp2S(1.fwdarw., corresponding to repeating 
units of the putative binding site for FGF-2. 
FGF antagonist activity of the glycosaminoglycans used in the present 
invention can be determined, for example, by the use of F32 cells (Ornitz, 
et al., Molec. Cell. Bio 12:240-247 (1992) (see, Example 1). 
The effective dosage for inhibition of angiogenesis in vivo, defined as 
inhibition of capillary endothelial cell proliferation and migration and 
blood vessel ingrowth, is extrapolated from in vitro inhibition assays. In 
vitro assays have been developed to screen for inhibition of angiogenesis. 
Events that are tested include proteolytic degradation of extracellular 
matrix and/or basement membrane, proliferation of endothelial cells, 
migration of endothelial cells, and capillary tube formation. The chick 
chriollantoic membrane assay (CAM), described by Taylor and Folkman, 
Nature (London), 297:307-312 (1982), is used to determine whether the 
compound is capable of inhibiting neovascularization in vivo. 
The effective dosage is dependent not only on the form of the 
glycosaminoglycan, but also on the method and means of delivery, which can 
be localized or systemic. For example, in some applications, as in the 
treatment of psoriasis or diabetic retinopathy, the inhibitor is delivered 
in a topical ophthalmic carrier. In other applications, as in the 
treatment of solid tumors, the inhibitor is delivered by means of a 
biodegradable, polymeric implant. 
The present invention further includes glycosaminoglycans formulated into a 
pharmaceutical composition. The pharmaceutical compositions of the 
invention include those suitable for oral, rectal, nasal, topical 
(including buccal and sublingual), vaginal or parenteral (including 
subcutaneous, intramuscular, intravenous and intradermal) administration. 
The formulations may conveniently be presented in unit dosage form, e.g., 
tablets and sustained release capsules, and in liposomes, and may be 
prepared by any methods well know in the art of pharmacy. See, for 
example, Remington's Pharmaceutical Sciences. 
Such preparative methods include the step of bringing into association with 
the molecule to be administered ingredients such as the carrier which 
constitutes one or more accessory ingredients. In general, the 
compositions are prepared by uniformly and intimately bringing into 
association the active ingredients with liquid carriers, liposomes or 
finely divided solid carriers or both, and then if necessary shaping the 
product. 
Compositions of the present invention suitable for oral administration may 
be presented as discrete units such as capsules, cachets or tablets each 
containing a predetermined amount of the active ingredient; as a powder or 
granules; as a solution or a suspension in an aqueous liquid or a 
non-aqueous liquid; or as an oil-in-water liquid emulsion or a 
water-in-oil liquid emulsion, or packed in liposomes and as a bolus, etc. 
A tablet may be made by compression or molding, optionally with one or more 
accessory ingredients. Compressed tablets may be prepared by compressing 
in a suitable machine the active ingredient in a free-flowing form such as 
a powder or granules, optionally mixed with a binder, lubricant, inert 
diluent, preservative, surface-active or dispersing agent. Molded tablets 
may be made by molding in a suitable machine a mixture of the powdered 
compound moistened with an inert liquid diluent. The tablets may 
optionally be coated or scored and may be formulated so as to provide slow 
or controlled release of the active ingredient therein. 
Compositions suitable for topical administration include lozenges 
comprising the ingredients in a flavored basis, usually sucrose and acacia 
or tragacanth; and pastilles comprising the active ingredient in an inert 
basis such as gelatin and glycerin, or sucrose and acacia. 
Compositions suitable for parenteral administration include aqueous and 
non-aqueous sterile injection solutions which may contain anti-oxidants, 
buffers, bacteriostats and solutes which render the formulation isotonic 
with the blood of the intended recipient; and aqueous and non-aqueous 
sterile suspensions which may include suspending agents and thickening 
agents. The formulations may be presented in unit-dose or multi-dose 
containers, for example, sealed ampules and vials, and may be stored in a 
freeze dried (lyophilized) condition requiring only the addition of the 
sterile liquid carrier, for example water for injections, immediately 
prior to use. Extemporaneous injection solutions and suspensions may be 
prepared from sterile powders, granules and tablets. 
Application of the pharmaceutical composition often will be local, so as to 
be administered at the site of interest. Various techniques can be used 
for providing the subject compositions at the site of interest, such as 
injection, use of catheters, trocars, projectiles, pluronic gel, stents, 
sustained drug release polymers or other device which provides for 
internal access. 
It will be appreciated that actual preferred amounts of a pharmaceutical 
composition used in a given therapy will vary depending upon the 
particular form being utilized, the particular compositions formulated, 
the mode of application, the particular site of administration, the 
patient's weight, general health, sex, etc., the particular indication 
being treated, etc. and other such factors that are recognized by those 
skilled in the art including the attendant physician or veterinarian. 
Optimal administration rates for a given protocol of administration can be 
readily determined by those skilled in the art using conventional dosage 
determination tests. 
The present invention is further illustrated by the following Examples. 
These Examples are provided to aid in the understanding of the invention 
and are not construed as a limitation thereof. 
EXAMPLE 1 
Material and Methods 
Glycosaminoglycans and their chemically modified derivatives. Heparin, low 
molecular weight heparin and heparan sulfate, sodium salts, were from 
procine intestinal mucosa and were obtained from Celsus Laboratories 
(Cincinnati, Ohio). The syndecan-I ectodomain was from NMMG cells and was 
quantified based on its heparin sulfate content (12). Acharan sulfate, 
sodium salt (12) and homogenous fully sulfated heparin oligosaccharides 
(13) were prepared and purified as described previously. N-deacetylation 
and N-sulfation of acharan sulfate followed literature methods (14). The 
structure of acharan sulfate and N-sulfoacharan sulfate relied on 
enzymatic disaccharide analysis, NMR spectroscopy and gradient PAGE for 
molecular weight determination (13). 
Interaction of glycosaminoglycans and their derivatives with FGF-2. 
Isothermal titration calorimetry was performed in 50 mM sodium phosphate 
buffer, pH 7.4, containing 100 mM sodium chloride at 25.degree. C. as 
previously described (15). 
Assay of FGF-2 mitogenic activity, FGF-2 mitogenicity assays were performed 
in F32 cells that express FGF-receptor 1 (FGF-R1)(16) but no detectable 
levels of heparan sulfate proteoglycans (17). Acharan sulfate and 
N-sulfoacharan sulfate were tested on F32 cells stimulated by 150 pM FGF-2 
in the presence of 10 ng/ml of heparin (inhibition of heparin-mediated 
activity) or in the absence of heparin (stimulation of FGF-2 activity). 
Tested glycosaminoglycans were added in concentrations ranging from 1-5000 
ng/ml. Proliferation was measured after 40 h incubation in 200 .mu.l 
medium (RPM1 1640, 10% heat-inactivated newborn calf serum) in the 
presence of a 1 .mu.Ci/well pulse of [.sup.3 H]-thymidine during the final 
6 h of the incubation. Radioactivity incorporated into DNA was quantified 
by scintillation counting, MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl 
tetrazolium bromide (Thiazole blue), used to determine cell viability, was 
from Sigma Chemical (St. Louis, Mo.). 
Results 
Preparation and structural characterization of glycosaminoglycan 
derivatives. Acharan sulfate was N-deacetylated and N-sulfated to obtain 
N-sulfoacharan sulfate (FIG. 1). The structure of acharan sulfate and 
N-sulfoacharan sulfate were assigned based on one and two dimensional 
.sup.1 H NMR spectroscopy (Table 1). The average molecular weight of 
acharan sulfate was 29,000 while the molecular weight of the N-sulfated 
acharan sulfate derivative was reduced to .about.8,000 as determined by 
gradient PAGE and confirmed by the sharpening of its NMR signals (see FIG. 
1). Treatment of acharan sulfate and N-sulfoacharan sulfate with heparin 
lyases I, II and III afforded the major (&gt;90%) disaccharide products, 
.DELTA.Uap2S(1.fwdarw.4)GlcNpAc (where .DELTA.Uap is 
4-deoxy-.alpha.-L-threo-hex-4-enopyranosyluronic acid) and 
.DELTA.Uap2S(1.fwdarw.4)GlcNpS, respectively, as determined by capillary 
electrophoresis (11). 
TABLE 1 
______________________________________ 
Assignment of .sup.1 H-NMR signals for glycosaminoglycan samples..sup.a 
Acharan sulfate.sup.b 
N-sulfoacharan sulfate 
Proton L-IdoAp2S D-GlcNpAc L-IdoAp2S 
D-GlcNpS 
______________________________________ 
H-1 5.t89 5.114 5.216 5.392 
H-2 4.345 4.020 4.331 3.374 
H-3 4.284 3.74 4.331 3.749 
H-4 4.027 3.47 4.130 3.838 
H-5 -- 3.867 4.889 3.946 
H-6 -- 3.87, 3.90 -- 3.84, 3.84 
N-Ac Methyl -- 2.083 -- -- 
______________________________________ 
a. 1HNMR was performed at 500 MHz in .sup.2 H.sub.2 O. Chemical shifts 
were determined relative to the internal standard, 3(trimethylsilyl) 
propioni acid1, sodium salt. 
b. S is used to designate sulfate and Ac to designate acetate. 
Determination of the FGF-2 binding affinity of glycosaminoglycans and their 
derivatives. Isothermal titration calorimetry was performed with heparin 
oligosaccharides, herapin, acharan sulfate and N-sulfoacharan sulfate. The 
heat of interaction (.DELTA.H) is directly measured in this experiment. 
From this heat of interaction the Kd, and n (stoichiometry) of interaction 
can be deduced. The minimum FGF-2 binding site is a tetra- or 
hexasaccharide. An n of 1.8 was observed for a dodecasaccharide suggesting 
a tetradecasaccharaide is required to bind two molecules of FGF-2 (FIG. 
2). Heparin and N-sulfoacharan sulfate bind FGF-2 most tightly, herapin 
oligosaccharides bind less well and acharan sulfate binds very poorly. The 
n value is consistent with the molecular weight of each ligand (Table 2). 
TABLE 2 
__________________________________________________________________________ 
Binding of glycosaminoglycans and their derivatives to FGF-2. 
Sample .DELTA.H (kcal/mole) 
K.sub.d (.mu.M) 
MW.sup.a 
n.sup.b 
avg. binding site size.sup.c 
Low molecular 
-13.4 0.036 
4,800 
3.6 
tetrasaccharide 
weight heparin 
N-Sulfoacharan sulfate -15.4 0.090 7,800 4.4 hexasacchar 
ide 
Acharan sulfate -15.1 4.5 29,000 13.4 octasaccharid 
e 
__________________________________________________________________________ 
a. Mwavg as determined by gradient polyacrylamide gel electrophoresis (11 
based on a repeating disaccharide unit (sodium salt) of mass 665, 563 and 
504 for heparin, Nsulfoacharan sulfate and acharan sulfate, respectively. 
b. n is the average number of FGF2 molecules occupying a single 
glycosaminoglycan chain. 
c. The size of oligosaccaride occupied by a single FGF2 molecule is 
2(MW/disaccharide unit mass)/n. 
Influence of glycosaminoglycans and their derivatives on FGF-2 mitogenic 
activity. The direct influence of acharan sulfate and its derivative on 
FGF-2 mitogenic activity was measured in the absence of added heparin. 
N-sulfoacharan sulfate, while binding FGF-2 with similar affinity as 
heparin, was about 150-fold less active than heparin on a weight basis, 
having about the same activity as the syndecan-1 ectodomain (FIG. 3A). In 
the presence of 10 ng/ml heparin (where [.sup.3 H]-thymidine incorporation 
would increase with increased heparin concentration), up to 1.5 .mu.g/ml 
of N-sulfoacharan failed to further stimulate FGF-2 mitogenicity (FIG. 
3B). In contrast, acharan sulfate markedly decreased the mitogenic 
activity in a concentration dependent manner, beginning at concentrations 
greater than that of the heparin. The effect of acharan sulfate on cell 
viability was assessed by measuring the formation of a chromogen from MTT, 
a tetrazolium dye that is converted solely by living cells (18). The assay 
showed no reduction in the number of living cells during 40 h incubation 
with the same acharan sulfate concentrations as used in the mitogenicity 
assays. Thus, the inhibition of mitogenicity is not due to toxicity 
leading to reduced cell number. 
The mitogenic activity of the FGF family of growth factors has been under 
extensive investigation (1,2,3). Heparin is known to enhance the activity 
of FGF-2 through its binding to this growth factor. The heparan sulfate 
chains of syndecan or glypican proteoglycans, while believed to be the 
endogenous molecules responsible for this activity, have considerably less 
mitogenic activity than heparin (6,19). Oligosaccharides prepared from 
heparin and heparan sulfate glycosaminoglycans bind FGF-2. The maximum 
FGF-2 binding site in heparin is a tetrasaccharide or hexasaccharide but 
neither enhances its mitogenic activity. It has been suggested that a 
larger heparin oligosaccharide that can promote dimerization of FGF-2 is 
required for mitogenic activity (20). The current study clearly 
demonstrates that a tetradecasaccharide is required for binding two FGF-2 
molecules (FIG. 2). A tetradecasaccharide fraction has been prepared from 
heparan sulfate both binds FGF-2 (6). 
The sequences of the oligosaccharides derived from heparin and from heparan 
sulfate that bind FGF-2 share a common feature. The FGF-2 binding heparin 
oligosaccharides contain the repeating sequence 
.fwdarw.4)GlcNp2S6S(1.fwdarw.4)IdoAp2S(1.fwdarw.(7,8) while the heparan 
sulfate oligosaccharides contain the repeating sequence 
.fwdarw.4)GlcNp2S(1.fwdarw.4)IdoAp2S(1.fwdarw.(4,5,6). The binding 
contribution of the 6-sulfate groups, commonly found in the glucosamine 
residues of heparin, and in the FGF-2 binding heparin oligosaccharides is 
unclear (21). 
Recently, acharan sulfate, a novel glycosaminoglycan of the structure 
.fwdarw.4)GlcNpAc(1.fwdarw.4)IdoAp2S(1.fwdarw., was isolated. Unlike the 
more structurally complex heparin or heparan sulfate, acharan sulfate 
contains a major (&gt;90%) repeating disaccharide unit, making it a 
relatively simple structure (11). Acharan sulfate's simple but unusual 
structure was chemically converted to a new derivative, N-sulfoacharan 
sulfate, containing the repeating saccharide present in heparan sulfate 
that binds FGF-2. The structure of N-sulfoacharan sulfate, 
.fwdarw.4)GlcNp2S(1.fwdarw.4)IdoAp2S(1.fwdarw., was established using NMR 
spectroscopy (FIG. 1). Although its average molecular weight was somewhat 
reduced, it gave a single disaccharide of the structure 
.DELTA.UA2S(1.fwdarw.4)GlcNS on treatment with heparin lyase I and II, 
consistent with its structure. 
Isothermal titration calorimetry has been used to measure the binding of 
heparin and heparin oligosaccharides to FGF-2 (8,10,15). Similar analysis 
showed that while N-sulfoacharan sulfate bound (K.sub.d of 0.09 .mu.M) 
with nearly the same affinity as heparin (K.sub.d of 0.036 .mu.M), acharan 
sulfate bound with much lower affinity (K.sub.d &gt;4 .mu.M)(Table 2). In 
addition, N-sulfoacharan sulfate tightly bound multiple FGF-2 molecules 
suggesting that it is capable of dimerizing FGF-2. These data confirm that 
the presence of 6-sulfate groups have little effect on the binding avidity 
of glycosaminoglycans to FGF-2 (6,21). 
Acharan sulfate and N-sulfoacharan sulfate have distinctly different 
effects on the mitogenicity of FGF-2 for F32 cells, a B-cell-derived cell 
line stably transfected with FGFR-1. Despite its high binding affinity for 
FGF-2, N-sulfoacharan sulfate had minimal mitogenic activity compared with 
that of heparin. This is in apparent contrast to both the high binding 
affinity and high mitogenic activity previously reported for the heparan 
sulfate-derived tetradecasaccharide fraction having a similar repeating 
structure (6). However the presence of minor levels of 6-sulfate groups in 
this fraction may account for the observed difference (6,21). The 
6-sulfate groups in the tetradecasaccharide fraction (or in heparin) may 
be important for enhancing its mitogenic activity presumably through their 
interaction with FGFR-1 (22). N-sulfoacharan sulfate had no discernible 
effect on FGF-2 mitogenicity induced by heparin. In contrast, acharan 
sulfate inhibited FGF-2 mitogenicity in the presence of heparin. This 
inhibition was seen at low GAG concentrations (IC.sub.50 of .about.400 
ng/ml in the presence of 10 ng/ml heparin). The inhibition was not due to 
either direct binding of FGF-2, because the growth factor-acharan sulfate 
interaction is very weak, or to toxicity that alters cell viability. 
Because acharan sulfate is a large anionic polysaccharide, it is not 
likely to exert its inhibitory effect by entering cells. However, acharan 
sulfate might bind to FGF-2 dimers, thought to be the growth factor's 
mitogenically active form (1-3, 8,9,20). The dimer shows a second 
glycosaminoglycan binding surface when stabilized by heparin (8,9,22). 
Interaction of acharan sulfate with this surface could account both for 
its low affinity for FGF-2 monomers (FIG. 2) and its inhibition of 
heparin-mediated FGF-2 mitogenicity (FIG. 3B). 
EXAMPLE 2 
Preparation of Low Molecular Weight Acharan Sulfate by Controlled 
Depolymerization of Acharan Sulfate with Heparin Lyase II 
Acharan sulfate (200 .mu.l at 1 mg/ml) in 50 mM sodium phosphate buffer, pH 
7.6 was treated with heparin lyase II (12 mU) at 30.degree. C. At various 
time points, the absorbance at 232 nm was measured and digestion was 
continued until the absorbance was constant (complete digestion). The 
percent reaction completion at each time point was calculated by dividing 
the absorbance at 232 by the absorbance measured at reaction completion. 
Acharan sulfate (3 ml of 1 mg/ml) in the same buffer was again digested 
with heparin lyase II. The digestion mixture heated at 100.degree. C. for 
3 min when the absorbance at 232 nm indicated the digestion was 55% 
complete. The partial digestion mixture was freeze-dried and reconstituted 
with 1.5 ml of distilled water and stored frozen for analysis by strong 
anion exchange-high performance liquid chromatography. The sample was 
injected on a 5 .mu.m Spherisorb strong anion exchange-high performance 
liquid chromatography column (Phase Separation, Norwalk, Conn.) of 
dimensions 2.5.times.25 cm equilibrated with water at pH 3.5 and eluted 
using a 120 min gradient from 0.0 to 1.8 M of NaCl pH 3.5 at a flow rate 
of 4.0 ml/min. The elution profile was monitored by absorbance at 232 nm 
at 0.5-1.5 absorbance unit full scale. This analysis showed an 
oligosaccharide mixture (Figure ?). The major components of this mixture 
(by weight) were oligosaccharides having 12 or more saccharide units. See, 
Determination of the Structure of Oligosaccharides Prepared from Acharan 
Sulfate, Y. S. Kim, M. Y. Ahn, S. J. Wu, D.-H. Kim, T. Toida, L. M. 
Teesch, Y. Park, G. Yu, J. Lin, R. J. Linhardt, Glycobiology, in press, 
1998. 
The disclosure of the references cited throughout the specification are 
incorporated herein by reference. 
The invention has been described in detail with reference to preferred 
embodiments thereof. However, it will be appreciated that those skilled in 
the art, upon consideration of this disclosure, may make modifications and 
improvements within the spirit and scope of the invention. 
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The invention has been described in detail with reference to preferred 
embodiments thereof. However, it will be appreciated that those skilled in 
the art, upon consideration of this disclosure, may make modifications and 
improvements within the spirit and scope of the invention.