This invention was supported in part by grant GM33063 from the National Institutes of Health. The U.S. Government may have rights in this invention.

IFTRQPUCTIPN Technical Field

This invention is directed to xylosides and their use as primers of heparan sulfate biosynthesis.

Background

Glycosaminoglycans are alternating polymers of hexosamine and alduronic acid which are found in sulfated forms. The members of the GAG family are classified by the nature of the hexosamine/alduronic repeating units. In chondroitin sulfate, the alduronic acid is primarily D-glucuronic acid (GlcA) and the hexosamine is acetylated 2-amino-2-deoxy-D-galactose (GalNAc) . In heparin and heparan sulfate, the hexosamine is primary acetylated and sulfated glucosamine (GlcNAc and GlcNS) and the alduronic acid is mostly L-iduronic acid (IdoA) in heparin and mostly D-glucuronic acid in heparan sulfate. There is some evidence that heparan sulfate is convertible to heparin by conversion of the GlcA residues to IdoA residues, which involves a change in chirality at the 5 position of the uronic acid residue.

Both heparan sulfate and heparin are important GAGs in physiological situations, as both are anticoagulants and are antiproliferative with respect to

smooth muscle cells. Elevated levels of these GAGs are therefore helpful in vivo in the context of preventing thrombosis and restenosis, as well as in other disorders that involve the proliferation of smooth muscle cells. The GAGs are naturally synthesized on protein substrates, to which a β-D-xylose unit has been attached (through an acetal linkage) via a hydroxy-containing side chain of an amino acid (usually serine) . In 1971, Okayama et al., Seikaσaku 4_3_:454 (1971) demonstrated the ability of p-nitrophenyl-β-D-xyloside to act as an artificial chain initiator for chondroitin sulfate biosynthesis. Since then, a number of publications have described β-D-xylosides and various analogs for use as artificial initiators of glycosaminoglycan chain synthesis both in culture and in vivo. See, for example, Esko et al., J. Biol. Chem. 262;12189-12195 (1987); Lugemwa et al., J. Biol. Chem. 266:6674-6677 (1991); Schwartz et al., Proc. Nat. Acad. Sci. USA 7l;4047-4051 (1974); Layne et al., Biochemistry 15:1268-1270; Kolset et al., Biochem. J. 265:637-645 (1990); Sobue it al., Biochem. J. 241:591-601 (1987); and Robinson et al., Biochem. J. 194:839-846 (1981) . Considerable differences exist in the structures of the aglycone portion of the synthesized molecule. For example, ethyl-β-D-xyloside, benzyl-β-D-xyloside, ethyl-β-D-thioxyloside, C-ethyl-β-D- xyloside and estradiol-β-D-xyloside have all been prepared.

Prior to work done in the laboratory of the present inventors in 1991, the β-D-xylosides previously used stimulated chondroitin sulfate (dermatan) synthesis while only weakly stimulating the more desirable heparan sulfate or heparin synthesis. The xylosides also tended to be poor primers even in those cells that only produce heparin or heparan sulfate. As reported in 1991 (Lugemwa, et al., OP cit) . estradiol-β-D-xyloside was

found to be an efficient primer for heparan sulfate biosynthesis. However, this selectivity was surprising, and no basis existed for predicting structures other than estrogens that would also favor heparan sulfate biosynthesis over chondroitin sulfate biosynthesis existed.

Thus, it remains an object of the present invention to provide further primers that favor heparan sulfate synthesis over competing GAG synthetic pathways.

SUMMARY OF THE INVENTION The present invention provides compounds that are β-D-xylosides of fused bicyclo aromatic molecules. The compounds of the invention thus have a first aromatic ring linked to xylose via a hydroxyl, thio, hydrocarbon, or amino substituent on the ring to form a β-D-xyloside (or equivalent) linkage. The fused bicyclo aromatic molecule also comprises a second aromatic ring fused to the first ring. Typical organic substituents can be present on the aromatic ring system. It has been found that these fused bicyclo aromatic β-D-xylosides, when used as primers of heparan sulfate biosynthesis in cultured animal cells, produce a significant amount of heparan sulfate compared to closely related molecules that do not contain a fused bicyclo aromatic moiety.

BRIEF DESCRIPTION OF THE DRAWINGS The invention will be better understood by reference to the following detailed description of the invention when considered in combination with the drawings that form part of this specification, wherein:

Figures 1-5 are graphs showing total ³⁵ S0 ₄ - labelled glycosaminoglycans, heparan sulfate, and chondroitin sulfate production for varying concentrations of five different primers of the invention.

Figure 6 is a graph showing reduction of restenosis in experimental animals in the presence of napthylxyloside, a primer of the invention, relative to use of carboxymethyl-cellulose, a control compound.

DESCRIPTION OF SPECIFIC EMBODIMENTS It has been found that fused bicyclo aromatic molecules coupled to xylose to form a β-D-xyloside (or a molecule with an equivalent linkage) function as primers for heparan sulfate biosynthesis.

Initial investigations that led to the present invention included the attempt to use various xylosides as primers of GAG biosynthesis. In these synthetic processes, the percent of heparan sulfate (HS) in the total GAG present was measured based on ³⁵ S0 ₄ incorporation under sulfate limited conditions. This method does not allow direct measurement of glycosaminoglycan mass since the amount of sulfate per chain and the length of chains varies between heparan sulfates and chondroitin sulfates. However, the relative efficacy of different xylosides to prime heparan sulfate can be measured in this way. As shown in Scheme 1, which lists a number of xylosides and shows their structures and the percent of heparan sulfate (HS) produced, only estradiol-β-D-xyloside produced as much as 50% of heparan sulfate (relative to total GAGs present) when fed to cells at 50 μM.

Estradiol-β-D-Xyloside

n-octyl-β-D-Xyloside Cholestβryl-β-D-Xyloside 5-10% HS 0% HS

p-nitrop enyl-β-D-Xylosidθ 4-mθthyiumbθliifβryl-β-D-Xyloside 5-10% HS 10-15% HS

Diet yistil estrol-β-D-Xyloside 30-35% HS

Scheme 1

Although estradiol-β-D-xyloside produced a satisfactory amount of heparan sulfate, estradiol has a number of physiological effects in animal cells and thus is not an ideal choice for inducing heparan sulfate biosynthesis either in vivo or even in cell culture. The inventors therefore investigated a number of different compounds, some of which are shown in Scheme 2. These compounds included monocyclic and bicyclic aglycone moieties attached to the xylose molecule, with both aromatic and non-aromatic ring systems being present. The best producers (in percentage) of heparan sulfate were xylosides formed from fused aromatic bicyclo molecules. Testing of additional molecules, including molecules with heteroatoms in the aromatic rings and analogs of xylosides with atoms other than oxygen forming the linkage between the xylose and aglycone, indicated that such molecules are capable of producing high relative amounts of heparan sulfate. Figures 1-5 show a series of bicyclo aromatic aglycone moieties used to form xylosides and the different percentages of heparan sulfate relative to chondroitin sulfate produced at different dose levels in a standard cell culture system that is described in more detail in the following examples.

Xyiosidθ (50 μM) 35 S-cpm/well β _{/o cs} o _{o HS}

phenyl-β-D-xyloside

4-n-butylp enyl-β-D-xyloside

tetrahydro-2-napt yl-β-D-xylosidθ

2-napt yl-β-D-xyloside

cis/τrans-decahydro-2-napthyl- β-D-xylosde

Scheme. -2

Accordingly, sufficient data is present to demonstrate that compounds comprising a β-D-xyloside of a fused bicyclo aromatic molecule are capable of acting as selective primers of heparan sulfate biosynthesis without having the physiological effects of estradiol, the molecule previously known to act as a heparan sulfate primer when present as a xyloside. Compounds of the invention comprise fused bicyclo aromatic molecules in which a first aromatic ring is linked to xylose via a hydroxyl, thio, hydrocarbon, or amino substituent to form a β-D-xyloside linkage or such a linkage in which the linking oxygen atom has been replaced with a carbon, sulfur, or nitrogen atom, with a second aromatic ring being fused to the first ring. In preferred embodiments, both the first and second rings are 5- or 6-membered rings.

Some substitutions can be present on the xylose moiety. For example, the ring oxygen can be replaced by sulfur (-S-), nitrogen (-NH-), or carbon (-CH ₂ -). The 2- hydroxyl can be methylated if desired. To avoid complexity of language, such xylose derivative are considered to be within the scope of the generic term "xylose." Suitability of any given substituent can be determined by the experimental procedures set out in the following examples.

Typical organic substituents can be present on the aromatic rings. For example, the first or second ring can be further substituted with a covalently attached substituent containing 1 to 10, preferably l to 5, atoms selected from the group consisting of C, N, 0, and S and sufficient atoms selected from the group consisting of H and halogen to form a covalently bound organic group that is not reactive at 37°C with water other than to participate in proton exchange or hydrolysis reactions that are compatible with

physiological systems (e.g., ionization of acidic and basic groups or hydrolysis of esters) . There is no particular limit on the number of substituents present, but 0, 1, 2, or 3 substituents are preferred, 0 or 1 being more preferred. The substituents can be on either of the aromatic rings but are preferred to be on the ring that does not contain the linkage to the xylose group. Examples of typical substituents include hydroxyl, carboxyl, ester, ether, amino, alkyl amino, nitro, cyano, halo, alkyl, haloalkyl, and haloalkoxyl substituents.

Substituents can be linear, branched, or cyclic and can be saturated or unsaturated. Preferred are small substituents such as those containing a single carbon atom (or no carbon atoms) along with other atoms as mentioned above, such as hydroxyl, carboxyl, methoxycarbonyl, carboxymethyl, methoxyl, amino, nitro, cyano, methylamino, methyl, and C-halogenated derivatives thereof.

A preferred compound is one having only hydrocarbon rings (such as naphthalene) , but heterocyclic rings can be presents. In preferred embodiments, the heterocyclic ring is selected from the group consisting of furan, pyrrole, thiophene, pyrazole, triazole, isoxazole, thiazole, isothiazole, pyridine, pyridazine, pyrimidine, pyrazine, and triazine. This heterocyclic ring is typically the second of the two aromatic rings, namely that ring which is not directly attached to the xylose moiety, but the first ring (or both rings) can be heterocyclic. Together, the first and second rings preferably form a molecule selected from the group consisting of naphthalene, benzofuran, isobenzofuran, benzothiofuran, isobenzothiofuran, indole, benzpyrazole, quinoline, isoquinoline, cinnoline, quinazole, naphthyridine, pyrido- [3,4-b] -pyridine, pyrido- [3,2-b] - pyridine, pyrido- [4,3-b] -pyridine, purine, anthracene,

and carbazole. Compounds that contain four or fewer 5- and/or 6-membered rings, and especially two aromatic rings.

A preferred group of compounds include those in which the compound ring system comprises a formula

wherein: Z is β-D-xylose;

L is -0-, -S-, -CH ₂ -, or -NH-; X is -CH-, -N-, -NH-, -0-, or -S-; m is 0, 1, or 2; n is 0, 1, 2, or 3; and m + n is 2 or 3; wherein X, m, and n, together with the remainder of said formula, are selected to form said second aromatic ring.

The following compounds are particularly preferred for use in the present invention: 2-naphthyl- β-D-xyloside, 5-indolyl-β-D-xyloside, 2-naphthylenethio- β-D-xyloside, 6-quinolinyl-β-D-xyloside, 1-naphthyl-β-D- xyloside, 6-quinolinylthio-β-D-xyloside, 6- benzothiopyran-β-D-xyloside, 6-benzopyran-β-D-xyloside, 5-indolyl-β-D-xyloside, 5-indenyl-β-D-xyloside, 9-phenanthryl-β-D-xyloside, and pyrenyl-β-D-xyloside. Although words used herein have their normal meanings when applied generally to the invention, the following definitions apply to preferred embodiments of the invention. A ring is "aromatic" if (1) atoms in the ring are located in a single plane and 4n + 2 pi electrons are present on the ring atoms or (2) the ring exhibits a ring current in the presence of a magnetic field; i.e., the electrons are delocalized.

A compound is a "derivative" of a first compound (as used herein for preferred embodiments) if the derivative compound is formed (or can be formed) by reaction of the first compound with another molecule or reagent so as to form a new compound either smaller or larger than the first compound while retaining at least part of the structure of the first compound.

A "moiety" is a part of a complex molecule that is derived from the indicated original named part. For example, the "aglycone moiety" of a xyloside is the part of the xyloside originally derived from the molecule to which the xylose has been attached (e,g., β-naphthol for 2-naphthyl-β-D-xyloside) .

A "substituent" is a moiety on an organic molecule that has replaced a hydrogen or other atom present in the named base molecule (e.g., a methyl substituent on one of the aromatic rings of naphthalene replaces one of the hydrogens present in a molecule of naphthalene) . A "β-D-xyloside" linkage preferably refers to a normal acetal linkage formed between D-xylose and an alcohol. However, for the sake of simplicity of language, any linkage that joins an aglycone via a hydroxyl, thio, hydrocarbon, or amino substituent to xylose at C-1 is considered to be a β-D-xyloside linkage as this term is used at its broadest. Such compounds are in fact analogs of xylosides in which the normal linkage of the aldehyde carbon of xylose to an alcohol oxygen of the aglycone is replaced by a linkage to a carbon, nitrogen, or sulfur of the aglycone (i.e., -0- is replaced by -CH ₂ -, -NH-, or -S-).

The xylosides of the invention can be synthesized from acetobromoxylose by the method of Koenigs et al., Ber 34:957-981 (1901). The alcohol, thio, or amino form of the fused bicyclo aromatic

compound is reacted with acetobromoxylose, and the products are isolated and identified by standard techniques. C-Alkyl xyloside analogs are prepared by reacting the corresponding alkyl Grignard reagent with a protected xylose molecule, such as tri-O-acetyl xylose. For detailed synthetic examples, see the previously cited references, particularly the experimental section of Sobue et al., op. cit.. which gives detailed synthetic processes for a number of different xylosides and analogs, including analogs in which the linking group is a sulfur atom or methylene group. The Sobue et al. publication also includes analytical data, such as NMR data, that will be useful when identifying products. Also see the examples set forth below. The compounds of the invention can be used either in vivo or in vitro. In vitro synthesis of heparan sulfate is carried out using animal cells, typically mammalian cells in culture, using normal culture conditions but with a compound of the invention incorporated into the growth medium. The optimum amount of xyloside to add to the culture medium for maximum weight or percentage production of a heparan sulfate will vary with the individual compound and the culture conditions. Typically the concentration will be at least l μM, with higher concentrations easily around 10 to 100 /xM producing higher total concentrations of GAG chains. Up to 1,000 μM of the xyloside have been found to be effective in some cases, although increasing dosages reduce the total amount of heparan sulfate produced in at least some cases. However, with at least some compounds such as 5-indolyl-β-D-xyloside, increasing amounts of the xyloside up to 1,000 μM, the highest dosage tested, continued to increase both the total amount of heparan sulfate and the relative percentage of heparan sulfate in the total GAG content.

Heparan sulfate is isolated from the reaction mixture using standard techniques, which are set forth in the references cited above and described further in the examples that follow. Compounds of the invention can also be used in vivo in contexts where the antiproliferative or antithrombosis activity of heparan sulfate is needed. Examples include prevention or reduction of thrombosis and/or restenosis in patients with cardiovascular problems. An example is the use of compounds of the invention to prevent restenosis in patients who have undergone angioplasty. Smooth muscle cell proliferation is a problem in angioplasty patients, and heparan sulfate has been shown to inhibit smooth muscle cell proliferation. Additionally, patients who are at risk for blood clot formation (patients who have previously suffered from stroke or a heart attack) can be treated with compounds of the invention to reduce the risk of further thrombosis. When administered to a patient, administration can be by any suitable route, such as intravenous, intramuscular, intraperitoneal, or the like. Oral administration is suitable for compounds that do not readily hydrolyze, such as C-alkyl analogues. For most administrations, compounds of the invention are administered in combination with a pharmaceutically acceptable carrier. Compounds of the invention are generally water soluble because of the presence of the xylose moiety, so that solutions (e.g., physiological saline or Ringer's solution) can be used. For transdermal or transmembrane administration, compositions which effect the transfer of active ingredients across the skin or across mucosal membranes are well known and can be used in the practice of the invention. Transdermal formulations are often

administered via skin patches; transmucosal is often accomplished by use of a suppository or through aerosol compositions.

Formulation of a composition of the invention into a form suitable for administration to humans or other mammals is standard technology now that the properties of the compounds of the invention are known. Accordingly, standard formulations can be used.

Pharmaceutical compositions containing a compound of the invention are prepared using standard techniques. The compound of the invention can be formulated into a solution or lyophilized powder for parenteral administration. Powders can be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carries prior to use. A liquid formulation generally comprises a buffered, isotonic, aqueous solution. Examples of suitable diluents are normal isotonic saline solution, standard 5% dextrose in water, and buffered sodium or ammonium acetate solutions. Such formulations are especially suitable for parenteral administration, but they can also be used for oral administration or contained in a metered doze inhaler or nebulizer for insufflation. Excipients such as polyvinylpyrrolidone, gelatine, hydroxy saline, acacia, polyethylene glycol, mannitol, sodium chloride, or sodium citrate, can be included in a pharmaceutical composition of the invention. Alternatively, compounds of the invention can encapsulated, tableted, or prepared in an emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers can be added to enhance or stabilize the composition or to facilitate preparation of the composition. Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, magnesium stearate, or stearic acid, talk, pectin, acacia, agar, or gelatin. Liquid

carriers include syrup, peanut oil, olive oil, saline, and water. The carrier can also include a sustained release material such as glyceryl monostearate or glyceryl distearate alone or with a wax. The amount of solid carrier varies with the particular use but preferably will be between 20mg to lg per dosage unit. The pharmaceutical preparations are made following conventional techniques of pharmacy such as milling, mixing, granulating, and compressing (when necessary for tablet forms) , or milling, mixing, and filling for hard gelatine capsule forms. When a liquid carrier is used, the preparation will usually be in a form of a syrup, elixir, emulsion, or an aqueous or non-aqueous suspension. Such liquid formulations can be administered directly p.o. or filled into a soft gelatine capsule.

For buccal administration, the compounds of the invention can also be combined with excipients such as cocoa butter, glycerine, gelatine, or polyethylene glucose.

When administered to a human, compositions are typically adjusted to give a cellular concentration of from l to 100 μM. This is typically accomplished by providing from 0.01 to 10 mg/kg per day. Unit pharmaceutical dosages (i.e., per capsule or tablet of 0.25 to 500 mg should provide a sufficient range of dosage possibilities for most purposes. However, determination of the optimum dosage is tailored to the individual patient as is well known in the art, and is generally adjusted by the medical practitioner. Thus, the dosage employed will depend on the condition of the patient, the manner of administration, the particular xyloside used, any known side effects of the aglycone, the manner of formulation (rapid release or slow release) , and the judgment of the attending physician.

Although the discussion above has addressed the issue of administration in the form of a single compound,

it will be apparent to those with skill in the art that both in vivo and in vitro techniques can be carried out using mixtures of the xylosides of the invention, either with each other in combination with other components used in the production of heparan sulfate.

The invention now being generally described, the same will be better understood by reference to the following detailed examples, which are provided for purposes of illustration only but which are not considered limiting unless so specified.

EXAMPLE 1 Synthesis of 2-naphthyl-β-D-xyloside 2-Naphthol (1.44 g, 10 mmol) is deprotonated with 1 mole equivalent of NaH in acetonitrile at room temperature. The resulting salt is reacted with 1.5 mole equivalent 2,3,4-triacetyl-α-D- xylopyranosyl bromide in situ to give the triacetyl-β-D- xylopyranoside of 2-naphthol. Removal of the acetyl groups with (MeOH:Et ₃ N:H ₂ 0, 2:1:1 v/v, at room temperature, 16 hours) and chromatographic purification furnishes 2-naphthyl-β-D-xyloside (up to 70% overall yield) .

This method works well with other aromatic hydroxyls. Different solvents, e.g. dioxane or tetrahydrofuran, can be used where acetonitrile is not suitable (with poorer yields) . The β-D-xylosides can also be purified by fractional crystallization or by flash chromatography on silicic acid (using ethyl acetate hexanes mixtures) as known in the art.

EXAMPLE 2 Glycosidation of Secondary Alcohols

Ten mol of the alcohol is dissolved in anhydrous dichloromethane (50mL) , 4A Molecular Sieves (MS, 2 g) and 2 mole equivalents of freshly prepared dry silver silicate. After stirring for 1 hour at room temperature in the dark, the mixture is cooled to -15°C. 2,3,4-Triacetyl-Q!-D-xylopyranosyl bromide (1.5 mole equivalent) is added, and the reaction mixture stirred at -15°C for 1 hour in darkness. The mixture is diluted with 50ml dichloromethane and filtered through Celite. The colorless filtrate is concentrated, and deacetylation is achieved as in Example 1.

Improved solubility of some alcohols in dichloromethane can be achieved by warming the solvent and using cadmium silicate instead of silver silicate. The products (yield 75%, overall) are purified by either fractional crystallization or flash chromatography on silicic acid using ethyl acetate/hexanes mixtures.

EXAMPLE 3 Glvcosidation of Primary Alcohols Ten mmol of 2,3,4-triacetyl-α-D- xylopyranosyl bromide is dissolved in 100ml anhydrous dichloromethane. To this is added the alcohol (1.2 mole equivalent) previously stirred with 4A MS for 24 hours at appropriate temperature, 1.5 mole equivalent freshly prepared "active" silver (I) carbonate, 0.5 g iodine, and 2 g 4A MS (in that order) . Drierite can be used in place of MS. After stirring for 16 hours in the dark, the reaction mixture is diluted with 100ml dichloromethane and filtered through a pad of Celite. The filtrate is concentrated to a syrup in vacuo.

Orthoester side-products are hydrolysed by dissolving the syrup in 100ml of 5mM sulfuric acid in

acetone: water 9:1 v/v, allowing it to stand for 30 minutes at room temperature, and then neutralizing with pyridine until a slight cloudiness appears. The mixture is again concentrated to a syrup. Deacetylation is achieved as in Example l. The β-D-xylopyranosides are purified by fractional crystallization or column chromatography on silica gel using ethyl acetate hexanes mixtures.

EXAMPLE 4

In Vitro Production of Heparan Sulfate Variation in glycosaminoglycan biosynthesis with concentration of various xylosides was determined in an in vitro system that is described in detail in Lugemwa et al., J. Biol. Chem. 266:6674-6677. See especially the detailed experimental procedures in this publication. In summary, approximately 2 x 10 ⁵ cells were plated in 60-mm diameter dishes. After 24 hours, the medium was changed to 1 ml of sulfate-free F- 12 growth medium containing. lOμCi of ³⁵ S0 ₄ and different concentrations of the xylosides shown in Figures 1-5 were added. The cells were incubated for 3 hours, and radioactive GAGs were isolated and identified, as shown in the Figures. In all cases, the proportion of heparan sulfate increased as the concentration of primer was increased. Recent experiments show that under these conditions the glycosaminoglycan chains are under- sulfated. However, inclusion of sulfate in the labeling medium does not change the order of efficacy of the various compounds, but decreases the apparent amount of heparan sulfate relative to chondroitin sulfate. Direct measurements of mass have not been made.

EEftMPLE ^~ Effect of 2-Naphthvl-β-D-xvloside In Vivo

Experiments were conducted to ascertain the beneficial effects of 2-naphthyl-β-D-xyloside on vascular disorders, and specifically the effects on restenosis caused by balloon catheterization. The experiments were done on rats essentially as described by Clowes A. W. et al., Lab. Invest. £i (1986) 295-303, and Guyton J.R. et al., Circ. Res. £ (1980) 625-634. Ten 4-week-old rats weighing about 350 grams were used for both the control and experimental group. Twenty-four hours before catheterization, control animals received carboxymethylcellulose (CMC) and the experimental group received 2-naphthyl-β-D-xyloside with CMC. Both were administered intraperitoneally in 1ml of fluid with or without an amount of 2-naphthyl-β-D- xyloside to give a dose of 1.75 mg/kg of rat body weight. Next, the animals were anaesthetized with a standard mixture of acepromzaine, xylazine, and ketamine, and the left common external carotid artery exposed in the neck through a midline incision, and denuded of endothelium using a 2-French balloon embolectomy catheter. The catheter was positioned in the artery, advanced to the aortic arch, then inflated and withdrawn three times to produce a consistent 3-cm segment of endothelial desquamation.

The animals were then injected daily for 14 days with either CMC or CMC with 2-naphthyl-β-D- xyloside. At the end of the 14-day period, all the control animals and 8 of the 2-naphthyl-β-D-xyloside treated animals were perfused with 2% glutaraldehyde, and the arteries were removed and immersed in 10% formalin in preparation for histologic examination. They were embedded and stained using standard histological procedures well known by those skilled in this art.

Next, the right and left common carotid arteries of both control and experimental animals were grossly examined for smooth muscle invasion into the intima, followed by two dimensional planimetric measurements to ascertain the area of the arterial lumen occluded by smooth muscle cell proliferation.

The two remaining experimental animals that were not sacrificed at the end of day 15 were later sacrificed on day 21 and processed for examination as described above.

Figure 6 shows the results of the planimetric measurements. Control animals that received CMC alone exhibited an intimal area of about 0.09/mm ₂ , compared to 0.07mm ² for 2-naphthyl-β-D-xyloside treated animals.

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.