Anionic saccharides for extraction of anti-angiogenic protein from cartilage

Anti-angiogenic protein composition can be prepared simply and efficiently by contacting cartilage particles containing the protein together with an electrolyte solution and an oligosaccharide bearing a minimum number of anionic substituents per sugar unit, to extract the anti-angiogenic protein from the cartilage particles and to form a complex of the oligosaccharide and the protein. The resultant anti-angiogenic complex can be used for therapeutic treatment of various angiogenic and related diseases.

This application derives priority from Provisional Patent Application 
60/004,022 filed Sep. 29, 1995. 
This invention is directed to compositions that inhibit the pathological 
growth of blood vessels in mammals, to methods of preparation of such 
compositions, and to the use of these compositions as therapeutic agents. 
BACKGROUND OF THE INVENTION 
1. Angiogenesis 
It was discovered in the 1970that the growth of tumors in mammals depends 
on the initiation, growth and continued formation of new capillary blood 
vessels (J. Folkman et al, "Isolation of a tumor factor responsible for 
angiogenesis", J. Exp. Med. 133, 275, 1971). The formation of these new 
blood vessels supports the growth of new tumor cells, just as the same 
process supports the development of an embryo or the healing of wounds. 
This process is known as neovascularization or angiogenesis. Agents that 
emanate from the tumor, for example, cause nearby blood vessels to produce 
new capillaries that grow towards and to the tumor and provide the 
nourishment necessary for its further growth. It is now known that the 
agents that are responsible for inducing neovascularization are growth 
factor proteins of the fibroblast growth factor (FGF) family. While a 
great many types of proteins exist and are involved in a large variety of 
different biological tasks, these growth factor proteins specifically 
induce proliferation and migration of endothelial cells, which are the 
cellular building blocks of blood capillaries and vessels. They are 
pro-angiogenic proteins, therefore. It is now also known that uncontrolled 
blood vessel growth is a phenomenon involved in pathologies other than in 
tumor growth. They are referred to as angiogenic diseases (see Battegay, 
E. J., "Angiogenisis: mechanistic insights, neovascular diseases, and 
therapeutic prospects", Review!, Journal of Molecular Medicine. 
73(7):333-46, 1995). They include for example diseases of the eye, such as 
corneal neovascularization, diabetic retinopathy and neovascular glaucoma; 
and other diseases such as hemangiomas, rheumatoid arthritis and 
psoriasis, among others. 
2. Anti-Angiogenic Agents 
The above discoveries led to a search for agents that might be used to 
inhibit angiogenesis. Cartilage, known to resist invasion by blood 
vessels, does not support the propagation of blood vessels (H. Brem et al, 
"Inhibition of tumor angiogenesis mediated by cartilage", J. Exp. Med, 
141, 427, 1975). Laborious experimental procedures of extraction of 
cartilage from rabbit, veal or shark have resulted in small concentrations 
of anti-angiogenic agent (R. Langer et al, Science 193, 70, 1976; R. 
Langer et al, Proc. Natl Acad. Sci. USA, 77, 4331, 1980; A. Lee et al, 
Science, 221, 1185, 1983) and have identified the substance to be a 
protein (M. A. Moses et al, Science 248, 1408, 1990). 
The methods for obtaining minute amounts of this anti-angiogenic material 
have been complex, involving multiple steps of extraction, purification, 
precipitation, separations involving ion exchange, desalting, 
chromatographic steps, filtrations, ultrafiltrations, dialysis and the 
like. 
The search for effective and affordable agents that would inhibit 
angiogenesis, that is for anti-angiogenic materials or formulations that 
would counteract the influence of pro-angiogenic proteins, has been 
continuing since these findings were made. Examples are the formulations 
of heparin with certain steroids (J. Folkman et al, Science 221, 719, 
1983), fumagillin (see U.S. Pat. No. 5,135,919 and the combination of 
sulfated cyclodextrins with specific steroids (Folkman et al, U.S. Pat. 
No. 5,019,562). The latter discovery poses significant background for the 
present invention, because it required formulation of the cyclodextrin 
agents with a steroidal or similar agent, without which the sulfated 
cyclodextrin would actually be pro-angiogenic, i.e., it would aggravate 
angiogenesis rather than inhibit it. Furthermore it was shown that the 
cyclodextrins having high anionic density will chemically complex 
pro-angiogenic proteinic growth factors (Y. Shing et al, Anal. Biochem. 
185, 108, 1990) and can be used to capture and separate the pro-angiogenic 
factors (Weisz et al, U.S. Pat. No. 5,183,809). 
Thus obtaining an effective naturally occurring anti-angiogenic agent from 
cartilage in useful concentration and quantity continues to be a highly 
desirable goal. 
SUMMARY OF THE INVENTION 
I have found that the anti-angiogenic protein present in natural cartilage 
can be extracted and concentrated, either as a solid composition or in 
soluble form, by a simple, inexpensive process. The extraction is done by 
intimately contacting solid particles of cartilage, a composition having a 
high intramolecular concentration of anion substituents and a solution of 
an electrolyte having a controlled concentration. The natural 
anti-angiogenic protein is transferred from the cartilage as a donor to 
the composition as a receptor. The resultant protein-receptor complex is 
separated from the cartilage particles to form an anti-angiogenic 
composition, either as a solid or solubilized in an aqueous solution, to 
form a concentrated anti-angiogenic protein product. 
The complexes themselves, or solutions containing the anti-angiogenic 
protein can be readily administered by various means to treat diseases 
requiring control of blood vessel formation.

DETAILED DESCRIPTION OF THE INVENTION 
3. The Method 
Animal cartilage is a unique source of protein, characterized by its 
anti-angiogenic capability to inhibit endothelial cell migration and 
proliferation and blood vessel generation, in contrast to the many known 
pro-angiogenic proteins, such as FGF and other growth factors that promote 
endothelial cell proliferation and migration. It is readily available from 
mammals, such as shark, in great abundance. It can be extracted from 
cartilage and transferred to a highly anionic acceptor substance in the 
presence of an electrolyte solution of a particular concentration. 
While this invention focuses mainly on extraction of anti-angiogenic 
protein from cartilage, it must be understood that other biological 
material containing such protein can be found, and the method taught by 
this invention may be used. However, other biological material generally 
has a large variety of proteins associated with it, including so-called 
growth factor proteins, which are pro-angiogenic, such as FGF. Thus 
additional steps of separation would be required to separate the desired 
anti-angiogenic agent. Thus the present method preferably is carried out 
with cartilage particles. 
In order to extract the anti-angiogenic protein from natural cartilage, 
finely divided particles of cartilage are contacted with a solution 
containing a saccharide having a minimum number of anionic substituents 
such as sulfates, and a particular concentration range of electrolyte, for 
a sufficient time to cause the transfer of substantial amounts of the 
anti-angiogenic protein from the cartilage to the anionic receptor. The 
electrolyte concentration is chosen to be high enough to displace the 
protein from the cartilage, while below the concentration at or above 
which the protein will not adhere or complex with the anionic receptor. 
The minimum number of anionic substituents on the receptor, preferably a 
saccharide, is chosen to assure strong protein absorption on the 
saccharide, while the type and concentration of the electrolyte is chosen 
to afford desorption of the protein from the cartilage particles. 
After intimate contact of the cartilage donor and the saccharide receptor 
in the electrolyte solution, preferably with agitation, has been provided 
for an adequate period of time, the cartilage particles are separated from 
the solution containing the extracted protein complexed to the saccharide. 
Any precipitated protein/saccharide complex may be obtained by settling or 
centrifuging and washing. any complex remaining in solution can be 
desalted by dialysis and concentrated by evaporation or lyophilation. 
In the preferred method of this invention, an anionic saccharide is chosen 
that is in solid particulate form. Thus, after the complexing procedure as 
described above, separation of the now solid saccharide/protein complex is 
easily achieved. For example, by choice of different ranges of particular 
size for the cartilage and for the saccharide particulates, separation is 
achieved by appropriately sized sieves. The choice of a solid protein 
acceptor is particularly advantageous because the anti-angiogenic protein 
product is now localized on the separated and easily washable solid, 
avoiding the need for costly techniques for removing the electrolyte and 
the need for considerable volumes of water to obtain a pure and 
concentrated protein product. 
In a typical preferred practice of this invention, cartilage particles, 
divided to a particle size of about 200-400 microns, and the receptor 
particles are stirred together in an electrolyte solution. A comparatively 
large difference in particle size between the cartilage and the receptor 
particles leads to easy separation of the particles after extracting and 
complexing using appropriately sized sieves. 
The particles of the receptor and animal cartilage are added to a dilute 
aqueous electrolyte solution, such as sodium chloride, at a concentration 
of electrolyte of 0.2-4 molar. An optimal concentration for monovalent 
anions is about 0.9 to 4 molar, whereas divalent ions are preferably 
employed as 0.2 to 1.0 molar solutions. Various electrolytes are useful 
herein, including salts of sodium, potassium, lithium, magnesium, calcium 
and the like. Thus although the chlorides are preferred, other salts can 
be used. The optimal concentration may vary with the particular receptor 
employed, e.g., an oligosaccharide, and the electrolyte employed. The 
suitability of the electrolyte solution concentration may be determined by 
adsorption of the receptor by a dye, as will be further explained 
hereinbelow, and should be less than that concentration at which 
appreciable dye adsorption occurs. Too low a concentration however will 
result in lengthy transfer times for the anti-angiogenic protein. 
The mixture of particles and electrolyte are stirred or agitated together 
for a time sufficient to transfer substantial and desired amounts of 
protein from the cartilage particles to the anionic receptor particles. 
The cartilage and receptor particles are then separated by methods such as 
controlled settling (large particles first), centrifugation or filtering 
using appropriate pore size screens, or by elutriation. When the particle 
sizes of the cartilage and the receptor have been chosen to be quite 
different, they can be separated readily using different size screens. 
It is also possible to employ sufficiently small particles of the anionic 
receptor solid, and sufficiently large particles of the cartilage (donor) 
solid, so that the former, in the manner of a fine dispersion, can 
circulate through the interstices of a retained bed of the cartilage 
solid. After a sufficient length of time of contact, the fine dispersion, 
now bearing complexed protein, can be "washed out" of the retained bed for 
collection. 
In another variant of the method, both types of solids are retained in 
separate beds by appropriately sized screens or membranes, and the 
electrolyte containing solution is circulated around and through both beds 
to afford contact and transfer of the protein. 
When the receptor used is a soluble compound, the resulting 
receptor-protein complex is obtained from solution. Excess electrolyte can 
be removed by ion exchange, dialysis, use of micromolecular filters and 
the like in known manner. The complex may be further concentrated or 
purified if desired. For example, the solution can be evaporated or freeze 
dried to remove water. The complex can be purified by molecular size 
chromatography to separate high molecular weight complexed products from 
non-complexed receptor, e.g., oligosaccharides, and other low molecular 
weight products. 
The complex may be used itself as a therapeutic agent, or the 
anti-angiogenic protein may be separated from the receptor or 
oligosaccharide by treatment with a strong (over 1 molar) electrolyte 
solution and further steps of separation. 
The use of solid particles of an oligosaccharide is preferred herein, 
however, because of the unique advantages and simplification of the 
separation procedures. When it is desired to produce the anti-angiogenic 
protein by itself, apart from its complex with a solid saccharide, this 
can be accomplished by treatment with a highly concentrated electrolyte, 
but the separation of the saccharide material thereafter is accomplished 
by simple mechanical manipulation, such as sieving, screening, settling, 
decanting and the like. 
The extraction and separations described above are preferably carried out 
at low temperature, to prevent damage to the extracted protein. Room 
temperature is preferred. However, a slightly lower or higher temperature, 
i.e. 15.degree.-50.degree. C., may enable extractions or de-salting to 
proceed more efficiently. 
Although the exact mechanism for the present method of preparation is not 
known with certainty, it is believed that the receptor oligosaccharides, 
having a minimum anionic concentration, offers a competitively stronger 
bonding or complexing strength for the anti-angiogenic protein than does 
the complexing partner that exists in natural cartilage; and the 
electrolyte having a required concentration in the transfer fluid, 
provides sufficient force to dissociate the cartilage-protein bonding 
without substantially interfering with the subsequent oligosaccharide 
bonding, thereby accelerating the rate of transfer of the protein in a 
practical period of time. 
4. The Protein-Receptor Compositions 
The compounds or materials useful in and characteristic of the invention 
comprise molecules with a high intra-molecular density of anionic 
substituents. They may be organic compounds, monomers such as suramin, or 
oligomers or polymers, synthetic or natural products, that have or are 
modified to have, the required high anionic density. I have found certain 
methods to determine the suitability, i.e., the existence of sufficient 
anionic density, to serve the methods of this invention. These methods are 
described further below. 
Saccharide-based materials are preferred because of their low cost, ready 
availability in quantity, and because of their general acceptability in 
biological systems. The oligosaccharides useful herein may be linear, 
branched chain or cyclic oligosaccharides. Hydroxy groups of such 
oligosaccharides are substituted with a minimum number of anions, such as 
sulfate, phosphate, sulfonate or carboxylate groups and the like. They can 
also include glycosaminoglycans (GAG), such as heparin or heparin 
derivatives including compositions referred to as low molecular weight 
heparins. The oligosaccharides useful herein have been reacted with or 
contain anions so that a minimum number of anionic substituents per sugar 
molecule is present. 
The preferred oligosaccharides are cyclodextrins, their isomers and 
homologs. These are low molecular weight cyclic oligosaccharides having at 
least 5 sugar units. Their structure is shown in FIGS. 1 and 2. FIG. 1 is 
a schematic illustration of the chemical structure of alapha, beta or 
gamma cyclodextrins. FIG. 2 is a three-dimensional view of the alpha, beta 
or gamma cyclodextrins useful herein. Generally the cyclodextrins 
preferred herein have 6-8 sugar units. These molecules, being cyclic, do 
not have terminal sugar groups, increasing their stability. Sulfated 
oligosaccharides are generally preferred. 
Anionic saccharides are generally water soluble. While it is within the 
scope of the present invention to employ the step of contacting the 
cartilage with a soluble species for protein extraction, sparsely soluble 
or substantially insoluble forms are preferred, as noted above, inasmuch 
as this provides substantial simplification of separation. For this 
purpose, the saccharides, or other highly anionic receptor compositions, 
are employed in the form of polymers or copolymers, or bound to solid 
surfaces through chemical or ionic linkage units. Methods of 
polymerization or cross-linking are well known in the chemical and polymer 
arts. For example, cyclodextrin particles have been obtained by known 
methods of polymerization; e.g., see N. Wiedenhof et al, Staerke, 21, 119, 
1969; cyclodextrin polymeric solids can be obtained in various particle 
sizes and properties, e.g., B. Zsadon et al, 1st Intern. Symp. on 
Cyclodextrin, 327, 1981; M. Komiyama et al, Polymer J. 18, 375, 1986; and 
U.S. Pat. No. 5,075,432. The critical anionic substituents can be added by 
treatment with a sulfating agent. This is shown in U.S. Pat. No. 
5,183,809, wherein the polymer was one of beta-cyclodextrin monomers 
linked through epichlorohydrin as the cross-linking agent, resulting in 
solid particles that are subsequently reacted with a sulfating agent such 
as chlorosulfonic acid, or trimethylamine/sulfate trioxide, so as to 
introduce that number of anionic sulfate groups to obtain the desired 
number of sulfate groups per monomer useful in the present method. While 
saccharides, and particularly cyclodextrin-derived materials are preferred 
for use in this invention, it is to be understood that other materials 
offering high anion density, such as resins and others, may be useful as 
protein receptors in the methods herein described. See for example U.S. 
Pat. No. 5,183,809 of Weisz et al. Dextran sulfate polymer can be 
similarly produced by linkage with epichlorohydrin, for example, prior to 
sulfating. Similarly, sparsely soluble or insoluble saccharides, already 
in particulate or solid form, such as starches or cellulose, may be 
employed after sulfation by suitable sulfating agents. Alternatively, 
copolymers of cyclodextrins with other materials, or linked to other 
solids, including cellulose, silica and other solid surfaces, may be 
employed, e.g., chlorodextrin/cellulose of Otta et al, Proc. 4th Inter. 
Symp. on Cyclodextrins, 139, 1988 or cyclodextrin/polyurethane, of Y. 
Mizobuchi et al, J. of Chromatography, 208, 35, 1981. 
5. The Anion Density for the Compositions of the Invention 
As discussed above, the degree of anionic substitution, preferably by 
sulfate ions, or more appropriately, their intramolecular density, is 
important. The anionic density required may vary somewhat with the size 
and structure of the saccharide molecule employed. For example, the 
preferred cyclodextrins will generally require a minimum of about 1.2 
sulfate groups/sugar unit. When sulfation is performed on only one side of 
the toroidal cyclodextrin molecule, they require a minimum of about 1.0 
anions/sugar unit. A linear oligomer of sugar units of more than about 
five sugar units, for example glucose units, requires a minimum number of 
about 1.4 or more anions per sugar unit. An example is dextran sulfate. 
Shorter chains of saccharides require a higher minimum, with a 
disaccharide requiring more than about 3.5 anions per sugar unit. Such a 
sulfated saccharide is exemplified by sucrose octasulfate. 
I have further found that the satisfactory achievement or presence of the 
minimum anionic density can be probed by their interactions with specific 
dyes. One is the shift in spectral color of the dye Azure A, known as 
metachromasia. Another test reaction makes use of the spectral color 
change or staining reaction of the dye Alcian Blue when reacted with the 
anion-substituted composition. In either or both of these tests, the 
candidate protein-acceptor composition for use herein should closely 
follow the like reactions of heparin. 
The test for metachromasia using the cationic dye Azure A has been 
described by Grant et al, "Metachromatic activity of heparin and heparin 
fragments", Anal. Biochem. 137, 25-32, 1984. This test is used to probe 
the adequacy of saccharide receptors for the purposes of this invention. 
Again, the amount of metachromasia should be similar to that of heparin. 
The test technique of Alcian Blue staining is described by Scott et al, 
"Differential staining of acid glycosaminoglycans (mucopolysaccharides) by 
Alcian Blue in salt solutions", Histochemie 5, 221-225, 1965; and by Snow 
et al, "Sulfated Glycosaminoglycans: A common constituent of all 
amyloids", Lab. Invest. 56, 120-123, 1987. It determines at what 
concentration an electrolyte such as magnesium chloride the test material 
will no longer stain. The technique is used in tissue histology. The most 
resistant substance, withstanding the highest electrolyte concentration, 
is heparin. Our criteria again is that the test results show that the 
protein acceptor is similar or equal to heparin. 
When the composition of the invention is a solid, staining can be observed 
directly. When the composition is soluble, a drop of its solution can be 
placed as a spot on paper or on a thin layer chromatography plate of 
alumina and dried. Staining can then be tested by a wetting solution of 
Alcian Blue dye with various strengths of electrolyte passed by the spot. 
These tests are useful because the above cationic dyes complex with the 
acceptor materials like proteins. The dyes are thus "models" for the 
complexation of proteins. 
6. The Range of Electrolyte Concentration 
As discussed hereinabove, there is a range of concentration of the 
electrolyte useful herein for the simultaneous contact with cartilage and 
with the protein receptor. The required or optimal electrolyte 
concentration for the present method will be between about 0.9 to 4.0 
molar for monovalent ions (Na, K, Li) and between about 0.2 and 1.0 molar 
for divalent ions (Ca, Mg). The optimal concentration may best be 
determined empirically, as it will depend on the combination of the 
particular oligosaccharide or other acceptor composition and the species 
of electrolyte used. A useful guide is obtained by the staining test as 
described above for Alcian Blue. The proper electrolyte concentration 
useful for the extraction method herein should be below that concentration 
at which appreciable dye staining occurs. However, lowering the 
electrolyte concentration will require a longer period of time to achieve 
appreciable transfer of protein. 
7. The Cartilage Protein 
The protein useful herein is an anti-angiogenic protein, characterized by 
its ability to inhibit endothelial cell migration and endothelial cell 
proliferation, in contrast to the many pro-angiogenic proteins such as FGF 
and other growth factors that promote endothelial cell proliferation and 
migration. Testing for the successful extraction or presence of the 
anti-angiogenic protein can be readily accomplished by independent in 
vitro tests in endothelial cell cultures, by virtue of successful 
reduction or inhibition of endothelial cell growth in culture. 
8. The Product Compositions 
The method of this invention results in the generation of novel and unique 
compositions with unusual utilities. The products include compositions 
that are complexed with anti-angiogenic proteins. The preferred complexes 
are anti-angiogenic proteins complexed with oligosaccharides. Preferably 
the complexes are solid particles or low solubility particles in aqueous 
solution. Although the anti-angiogenic protein may also be obtained per se 
by the further steps of de-complexation and separation, these complexed 
compositions are themselves highly useful therapeutic agents. When such a 
complexed composition is introduced to an environment free of the protein 
of the complex, the complexed anti-angiogenic protein will slowly leak out 
by de-complexation, as required by the laws of equilibrium. Further, if 
the biological environment contains other proteins, including FGF or other 
pro-angiogenic proteins, they will compete for complexation and thereby 
accelerate desorption of more of the anti-angiogenic protein into that 
biological environment. The complexed agent of this invention constitutes 
a pharmaceutical, anti-angiogenic agent. It is also a vehicle for delivery 
of the active agent which can be targeted to the biological environment 
where it is needed. The complexing agent, such as an oligosaccharide and 
preferably a sulfated cyclodextrin, complexed with the anti-angiogenic 
protein, is targeted to the biological site where it is needed, and slow 
delivery of the anti-angiogenic protein by equilibrium desorption proceeds 
at the site of introduction. 
The products of the present invention can be administered for therapeutic 
purposes in known manner. The complexed product per se is a solid and it 
may be also used in the form of a suspension. The extracted product can be 
used in solution as a dilute aqueous or salt solution. 
For treatment of tumors, the compositions of the invention inhibit the 
growth of tumors and thus can be administered topically as a solution, 
e.g., intravenously or sub-cutaneously. Compositions of the invention in 
the form of solutions or dispersions of solid particles can be delivered 
in the form of an ointment or gel for treatment of skin carcinomas. Fluid 
delivery of soluble compositions can be used to deliver the composition to 
tissue surrounding a tumor. 
For treatment of arthritic joints, the compositions of the invention can be 
applied topically in solution or as a dispersion in the form of a gel or 
cream. Solutions of the invention may be injected into the sinovial fluid 
around the affected joint. 
For treatment of diseases of the eye, soluble compositions of the invention 
may be formulated as eye drops for direct administration to the eye. 
For treatment of gastrointestinal tract diseases, solid compositions of the 
invention can be administered orally in a carrier that will survive 
stomach acidity, and then be absorbed into the blood plasma. 
For treatment of bronchial or pulmonary diseases, the dry or wet 
compositions of the invention can be nebulized and inhaled, or made into a 
spray solution. 
The invention will be further illustrated by the following examples. 
However, the invention is not meant to be limited to the details described 
therein. 
EXAMPLE 1 
A sample of beta=cyclodextrin was sulfated using trimethylamine/SO.sub.3 
complex to obtain a mixture of beta-cyclodextrin sulfates (CDS). The 
mixture was passed over a sephadex column to yield separate fractions 
containing cyclodextrin with varying average sulfate content, which then 
was determined by elemental analysis. The following materials were 
obtained: CDS with about 7 sulfate groups, CDS-7; and beta-cyclodextrin 
with about fourteen sulfate groups, CDS-14. After impregnating a spot on a 
thin layer chromatography plate of alumina with a drop of solution of each 
agent which was then dried, the staining ability of that spot with a 
solution of Alcian blue dye with various strengths of electrolyte 
(MgCl.sub.2) was tested. An unsulfated beta-cyclodextrin (CD) was also 
tested, as was heparin. Table I summarizes the data obtained. 
TABLE I 
______________________________________ 
Saccharide Concentration 
No Staining at 
Compound mg/ml MgCl.sub.2 Concentrations: 
______________________________________ 
CD 0.8 any 
CDS-7 0.8 any 
CDS-7 2.4 any 
CDS-14 0.8 &gt;1.0 molar 
Heparin Control 
0.8 &gt;1.0 molar 
______________________________________ 
Thus CDS-14 stains as strongly as heparin. No staining was obtained with 
CDS-7, even at a three-fold higher concentration. 
Thus a cyclodextrin sulfate with about 14 sulfate groups is a capable 
protein absorber, and demonstrates the need for a minimum number of 
sulfate substituents. 
EXAMPLE 2 
Another series of beta-cyclodextrin sulfates having various amounts of 
sulfate substitution were obtained by sulfating the cyclodextrin and 
separating the products. After sulfate analysis, they were subjected to 
the metachromasia test with the dye Azure A described hereinabove. The 
results are summarized below in Table II. 
TABLE II 
______________________________________ 
Number of Sulfate Substituents 
Metachromatic 
per cyclodextrin Activity 
______________________________________ 
0 0 
2.3 0.1 
4.0 0.1 
6.0 0.15 
10.4 0.95 
14 0.98 
15.8 0.97 
heparins 0.9-1.0 
______________________________________ 
The results identify samples having adequate complexing capability for the 
methods of this invention. They are the saccharides having a sulfate 
density greater than 6, and preferably 10 or more. 
EXAMPLE 3 
This example demonstrates the capability of the receptor oligosaccharide 
CDS-14 for its successful capability to complex a protein. 
When two substances are mixed and placed into an electrical conductivity 
device in varying proportion with another substance, the conductivity is 
known to be the linear average of the conductivity of each of the two 
substances alone. However, if the substances interact by complex 
formation, a strong deviation from that average will be noted. 
Using a standard conductivity probe and a total concentration of protamine 
and of CDS-14 of 4 mg/ml, the conductivity of protamine (100%) was 
observed to be 1280 micromhos. The conductivity of CDS-14 was 880 
micromhos. Thus one would expect that a 50:50 mixture of the two 
components would have a conductivity half-way in between, or 1080 
micromhos, if no complexing occurs. The observed conductivity however was 
1250 micromhos, showing a strong electrostatic complexing of CDS-14 with 
the protein. 
EXAMPLE 4 
This example illustrates the extraction method of this invention. 
Particles of cyclodextrin sulfate polymer (CSP) are dried under vacuum and 
ground to a particle size of about 3-10 microns. Cleaned animal cartilage 
having a particle size of about 1 mm (100 grams) and 2 grams of CSP as 
above are added to an aqueous solution of 0.6 molar NaCl. The particles 
are agitated for 60 hours at 30.degree. C. 
The larger cartilage particles are allowed to settle by sedimentation 
during mild stirring. 
The CSP-containing fluid is decanted, with additional CSP washed from the 
bed of sieve supported CAC. The CSP-containing fluid was filtered and the 
solid CSP product, now associated with extracted and complexed protein, is 
collected. This product is dispersed in aqueous nutrient and is added to 
an endothelial cell culture. The CSP product reduces endothelial growth 
rate in that culture compared to a control culture. 
EXAMPLE 5 
The protein is extracted from a sample of the CSP product of Example 4 by 
exposing the product to a solution of 4M NaCl for 12 hours and separating 
from the solution by filtration. The solution is dialyzed and lyophilized 
to remove the electrolyte and to obtain a concentrated protein solution. A 
small sample of the protein product is added to an in vitro cell culture 
of endothelial cells. The protein strongly inhibits cell proliferation. 
EXAMPLE 6 
Part A 
Dextran sulfate (DS8000), heavily sulfated gamma-cyclodextrin and maltosyl 
beta-cyclodextrin sulfate were exposed to Azure A dye solution. They 
exhibited strong metachromasia. 
Each sample was analyzed and was found to have a sulfur content greater 
than about 1.4 sulfate groups per sugar unit. 
Part B 
Unsulfated dextran, methylated cyclodextrin and hydroxypropyl cyclodextrin 
were subjected to the same test and did not exhibit metachromasia under 
the same conditions. 
Thus only the three heavily sulfated saccharides were shown to be operable 
as protein receptors in the method of the present invention. 
EXAMPLE 7 
100 Grams of CAC particles are immersed in 150 ml of an aqueous solution of 
0.6M NaCl electrolyte and 1 g of dissolved beta-cyclodextrin having about 
12 sulfate substituents per molecule of CDS is added. The mixture is 
agitated for 48 hours. 
The solution, having dissolved CDS with complexed protein, is separated by 
filtration, the electrolyte is removed by dialysis, and the volume is 
reduced by lyophilization. 
Samples of the product contain anti-angiogenic protein. The samples added 
to endothelial cell cultures inhibit cell growth compared to a control 
culture. 
EXAMPLE 8 
This example demonstrates the utility of a saccharide/protein complex of 
the invention to be useful as a therapeutic vehicle for delivery of the 
protein, i.e., to absorb the protein and subsequently to desorb the 
protein to a fluid that is devoid of that protein. 
A sample of CDS was immersed in a weak solution of Azure A and of bFGF 
protein to observe the time course of sorption. Upon reaching a saturated 
state, the polymer was then exposed to a clear aqueous phase and the 
progress of desorption was observed. 
The results are shown in FIGS. 3A and 3B. FIG. 3A shows the ability of the 
sulfated oligosaccharide, once complexed by absorption, to slowly and 
steadily deliver its charge to an aqueous environment by desorption 
(decomplexation) as shown in FIG. 3B. 
Although the present compositions and methods of treatment have been 
described in terms of specific embodiments, the invention is not meant to 
be limited except by the appended claims.