CA2193941A1 - Use of moieties for binding to hyaluronan and icam-1 - Google Patents

Abstract

The use of an effective amount of a compound having the general formula R1R2N(CH2)mNR3R4 is provided for the inhibition of hyaluronan, wherein R1 and R2 are selected from H and (see fig. I) and R3 and R4 are selected from H, (see fig. II) , (see fig. III) and (see fig. IV) and wherein m is an integer from 1-10.

Description

-1- 21 93~41 TITLE OF INVENTION
Use of moieties for binding to hyaluronan and ICAM-1.

SCOPE OF INVENTION
This invention relates to the use of moieties for binding to hyaluronan and ICAM-1. In some embodiments these moieties contain diaminohexane (such as 1,6-hexanediamine) and hexamethylene bis-acetamide.

BACKGROUND OF THE INVENTION
The human body, when reacting to a condition, in some instances, causes more damage. For example, when the body responds with an inflammatory process, more damage will result. Intercellular adhesion molecule-1 (ICAM-1) is a glycoprotein predominantly expressed on vascular endothelium, and mediates neutrophil and macrophage extravasation in inflammatory states. The elimination of the action of ICAM-1 from the inflammatory process is therefore desirable. For example, in reperfusion injury arising from strokes or cardiac infarction, the leukocytes stick to the arterial walls causing further damage. In transplants of kidneys, liver, etc., blood flow stops to specific areas, oxygen is reduced, then restarted, and the leukocytes penetrate into the tissue causing damage. Therefore, it is desirable to inhibit any ischemic damage. One method is to inhibit/block the action of ICAM-1.
It is therefore an object of this invention to inhibit/reduce, as much as possible, the effects of ICAM-1 in, for example, the inflammatory process.
It is also important to separate hyaluronic acid (hyaluronan) from other compounds and components with which it is combined. In this regard, this invention provides a method, and compounds containing molecules which may be used in such methods to separate the hyaluronan. This involves the combination of a compound containing moiety which reacts with hyaluronan ~' 2!93q41 -which then enables the separation of the combined compound containing the moiety component and hyaluronan from other components.
The moiety in the compound may also be used to inhibit/block the action of ICAM-1 and hyaluronan in the body by administering a dosage amount of a 5 compound containing a moiety which in the body combines with the ICAM-1 and/or hyaluronan, inhibiting its further action in the body. The moiety therebyinactivates ICAM-1 and hyaluronan in the body.
It is therefore an object of the invention to provide compounds containing moieties which accomplishes the above which includes diamino hexane or 10 hexamethylene bis-acetamide in the moiety (in effective dosage amounts) whichis specific for hyaluronan and ICAM-1. The moiety may also be used to bind hyaluronan to drugs for more effective administration of the drugs.
Further and other objects of the invention will be realized by those skilled in the art from the following summary of invention and detailed description of 15 embodiments.

SUMMARY OF INVENTION
According to one aspect of the invention, a non-toxic compound in a non-toxic effective amount of the formula RR1-N-(CH2)n-NR2R3 or a compound 20 containing a moiety of the formula 2 2 can be administered to a mammal (e.g. human) in an effective non-toxic therapeutic amount to block the action of hyaluronan and ICAM-1 in the mammal by, for example, reacting with the compound or compounds containing moiety. R, R1, R2 and R3 may be 'R b' C--CH3 or C (CH2)nCH3 while n=1, 2 or 3 or one of R, R1, R2 or 25 R3 may be a drug combined with the moiety.

R--N(CH2)nNR2 Where the non-toxic compound contains at least one of 1~l f' R and R2 may C CH3 or --C(CH2)nCH3 Thus, according to another aspect of the invention, the said non-toxic 5 c~mpound may have the formula R4-NH--(CH2)n--NH--R5 h R4 or Rs are each H or 6 where R6 is CnH2n+1 where 1<n<5. Where R3 is a drug it may be selected from the following although the list is not exhaustive.
R= NSAIDS such as acetic acid types and propionic acid types (especially ibuprofen) anti-inflammatory gold compounds such as auranofin (Merck Index 10th Ed. no. 882) 4-aminoquinoline type drugs such as chloroquine Colchicine Vinca alkaloids such as: Vinblastine (Merck Index 10th Ed. no. 9784) Vinblastine (Merck Index 10th Ed. no. 9788) Vinblastine (Merck Index 10th Ed. no. 9789) Histamine H1-antagonist drugs of the general structure:

Ar2/ f ~ ~Rl x=c N

20 Cyclosporin Glucocorticoid type agents Steroids Anabolic steroids such as nandrolone (Merck Index 10th Ed. no. 6211) Oestrogen type drugs such as stilboestrol Anti-androgen drugs such as cyproterone Contraceptive drugs Muscarinic antagonists such as atropine, homoatropine, cyclopentolate, tropicamide Medium duration anticholinesterase drugs such as physostigmine Histamine H2-receptor antagonists Adrenoreceptor antagonists such as propranolol, ergotamine, alprenotol, practolol, metoprolol.
Depending on the compound suitable dosages will be in the order of 1 to 10,000 micrograms per 70kg person which is adjusted proportionately up or down depending on the weight of the person.
According to another aspect of the invention, the compound or compound containing the moiety or combined drug and moiety can be administered to a mammal (e.g. human) in a suitable effective non-toxic therapeutic amount to inhibit the effect of ICAM-1 to inhibit the action (effect) of ICAM-1 in the inflammatory process in the mammal.
According to another aspect of the invention, the compound or compound with moiety may be used to purify hyaluronan by combining the compound or compound with the moiety with a solution comprising hyaluronan to permit its binding with the hyaluronan, thereafter recovering the bound combination of the moiety and hyaluronan and thereafter releasing the compound with moiety or compound from the hyaluronan and recovering the pure hyaluronan. The releasing of the hyaluronan from the compound or compound containing the moiety may be accomplished by dialysis as I have found that hyaluronic acid teaches very slowly from columns where it is non-covalently bound. Thus dialysis through a low molecular weight cut-off membrane should be a good way of separating 1,6-diaminohexane or hexamethylene bis-acetamide from HA. Electroelution and electrodialysis as known methods could also be similarly used.

2i~3941 -The non-toxic effective amount of the compound or compound containing the moiety may be administered by any suitable manner such as intravenously in saline and therefore may be solubilized in any suitable amount.A suitable effective non-toxic amount of the compound or compound 5 containing moiety for administration to a mammal is between about 1 to 10,000',1g/70kg person preferably between about 1000 to 6000~g (1 to 6mg)/70kg person. The dosage amount will be varied directly with the weight of the patient.
Thus, a 105kg person will be given 1.5 times the dosage amount a 70kg person will be given. The calculation of the amount does not include the amount of any 10 compounds, such as drugs, with which it is combined.
One such suitable compound is hexamethylene-bisacetamide (HMBA). A
discussion of its toxicity in rats has been described in an article entitled:
"Invest New Drugs 3:263-272 (1985) Distribution, elimination, metabolism and bioavailability of hexamethylenebisacetamide in rats.

Litterst CL, Roth JS, Kelley JA

Hexamethylenebisacetamide (HMBA), an in vitro differentiating agent, was studied for its pharmacodynamic actions in animals.
Plasma stability, organ distribution, excretion, oral bioavailability, and estimates of pharmacokinetic parameters and acute lethality were determined in rats. The single dose intraperitoneal LD50 was greater than 3000mg/kg in both mice and rats. The drug was stable in plasma from several different species during an 8h in vitro incubation at 37 degrees C. Following a single intravenous (iv) bolus injection (lOOOmg/kg) to rats, HMBA was removed from the plasma with a half time of 2.2 +/- 0.5h, and 65+/- 8% of the dose was excreted unchanged in the urine during the first 24h after dosing.
During an 8h iv infusion, plasma concentrations of 4mM were easily maintained with no apparent adverse effects. Drug was uniformly distributed, with highest concentrations found in thymus, kidney, liver, and lymph node throughout the first 24h after a single iv bolus dose. In vivo metabolism was very small, and the presence of the apparent metabolites was undetectable until 48h after iv administration. Oral bioavailability was good (32%), with peak plasma concentrations of 2mM achieved one hour after oral administration. After oral dosing urinary excretion and plasma decay were comparable to similar data obtained after iv dosing."

Other suitable compounds and moieties may be as follows:

(i) ~0.881mgbound \ INlH 1~l R--O--C--NH--(CH2)6-NH--C--CH3 of 0.950mg applied (ii)~0.829mg bound ~ R--O--CH2-CH--CH2-NH--(CH2)6--NH C CH3 ~of 0.950mg appliedJ
IOH
(iii)--R--O--CH2-CH--CH2-NH--(CH2)6 -NH3 (iv) hexamethylene bis-acetamide (HMBA) LD5o>lgm) CH3-cNH(cH2)6NHccH3 -belongs to class called Il "polar-planar compounds"
O or "hybrid polar compounds"

R

(v) --R-NH(CH2)6NHCCH3 binds hyaluronic acid where R is a drug binding due to (vi) --NH2(CH2)6NH2 ; positively charged amine on the ethyl amine (vii)~0.373mg bound ~ I /
O--C--NH--(CH2)--NH2 0.950mg applied - moiety above can bind drugs to hyaluronic acid They all have the general formula R1R2N(CH2)mNR3R4 1~l wherein in some embodiments R1=R2=H, C--CH3 fi~ ~I H Nl H
R3=R4=H, C--CH3, 0--CH2-CH--CH3, C--ORs The separation of the moiety or compound from hyaluronic acid can be 5 accompanied by the use of dialysis through a low molecular weight cut-off membrane or electroelution and electrodialysis in known methods.
The invention will now be illustrated with reference to the following examples:
Intercellular adhesion molecule-1 (ICAM-1) is a glycoprotein 10 predominantly expressed on vascular endothelium, and mediates neutrophil and macrophage extravasation in inflammatory states. Our laboratory has previously reported that ICAM-1 is a cell surface receptor for the polysaccharide hyaluronan (HA) (McCourt et al., (1994) J. Biol. Chem. 269, 30081-4). This finding was based on studies using HA oligosaccharides to elute ICAM-1 from columns 15 of HA attached to Sepharose 4B via a 1,6-diamino-hexane (DAH) based linker. In this report, we show that considerable amounts of ICAM-1 also bind the same linker in the absence of coupled HA, and that ICAM-1 does not bind HA attached -8- 2 I q394 1 to Sepharose 4B via a shorter linker, which suggests that ICAM-1 may have simply been displaced, and not specifically eluted, by HA from HA-DAH-Sepharose. We have also used HA attached to Sepharose 4B via the shorter ethylene diamine-based linker to purify HA binding proteins form 125I surface 5 labeled Triton X-100 solubilized liver endothelial cells. Bands of (approximately) 400, 200 and 84 kDa were eluted from this column with HA, although the 84 kDa species was also similarly eluted from the control resin. Immunoblots of this material were negative for ICAM-1.
Abbreviations used: HA, hyaluronan; LEC, liver endothelial cell(s);
10 HARLEC, HA receptor on liver endothelial cells; ICAM-1, intercellular adhesion molecule-1; EDC, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide; EDA, ethylenediamine; DAH, 1,6-diaminohexane; control-EDA- and control-DAH-Sepharose, 1-amino-2-acetoamidoethane- and 1-amino-6-acetoamidohexane Sepharose 4B; HA-EDA- and HA-DAH-Sepharose, HA-ethylenediamine- and 15 HA-1,6-diaminohexane-Sepharose 4B; P-HA-DAH- and P-control-DAH-Sepharose, Pharmacia-HA- and -control-DAH Sepharose 4B; AH-Sepharose 4B, aminoethane-Sepharose 4B; AE-Sepharose, aminoethane-Sepharose 4B; EAH-Sepharose, epoxy-linked aminohexane-Sepharose 4B; control-EDAH-Sepharose, epoxy-linked 1-amino-6-acetoamidohexane Sepharose 4B; HMBA, N,N'-20 hexamethylene-bis-acetamide; HA-ASD,2(p-azidosalicylamido)ethyl-1,3'-dithiopropionate-HA;PBS, phosphate buffered saline; PBS-TB, PBS containing 0.1% (v/v) Triton X-100 and 10mM benzamidine.
Intracellular adhesion molecule-1 (ICAM-1; CD54) is a glycoprotein expressed predominantly on vascular endothelium [1]. It is upregulated in 25 inflammatory states [2, 3] and mediates cell-cell adhesion and neutrophil/macrophage extravasation as a counter-receptor for lymphocyte function associated -1(LFA-1)(4,5) and the macrophage-associated Mac-1 [6].
Hyaluronan (hyaluronic acid; HA) is a polysaccharide found predominantly in soft connective tissue and has a wide range of functions including space filling, lubrication and providing a hydrated matrix through which cells can migrate [7-9]. HA enters the blood via the lymph and is rapidly taken up by liver endothelial cells (LEC) via high affinity receptors that also recognize chondroitin sulphate [10]. In many inflammatory states and in certain malignancies the 5 serum level of hyaluronan is elevated [11]. Both ICAM-1 and HA play pivotal roles in cell adhesion and migration.
Previous studies in this laboratory have identified an 85-100 kDa protein on the surface of rate LEC, with an apparent affinity to HA [12] attached via a 1,6 diaminohexane based linker to Sepharose (HA-DAH Sepharose) [13]. Fab 10 fragments of a rabbit polyclonal antibody raised against the protein could inhibit HA binding to both LEC membranes and LEC in culture, though in the latter case pre-immune Fab fragments also caused a significant inhibition of HA binding.
In immunohistochemical studies using this antibody, denoted as anti-HARLEC
(anti-HA receptor on liver endothelial cells), it was found that, with the 15 exception of rat liver endothelium and the capillaries of the kidney, hyaluronidase pretreatment of sections was required for significant staining [14, 15]. The same anti-HARLEC antibody provided the means to follow the large scale purification of an approximately 90 kDa protein from whole rat liver, withthe HA oligosaccharide elution of the protein from the above HA-DAH resin as 20 the penultimate affinity chromatography step. Sequencing of tryptic fragments of the 90 kDa species [16] and subsequent immunoblotting revealed that this proteinwas closely related or identical to ICAM-1, a potentially interesting finding in that it pointed to a role for HA in ICAM-1 mediated cell adhesion in inflammatory states. However, in this report, we show that the basis for demonstrating that 25 ICAM-1 interacts with HA, namely its elution from HA-DAH-Sepharose with HA oligosaccharides, must now be considered in the light of the present findingswhich reveal firstly that equivalent amounts of ICAM-1 in fact bind both the control DAH- and HA-DAH-Sepharose, secondly that HA oligosaccharides also has an affinity both resins and thirdly that ICAM-1 does not bind HA attached to

- 2 1 9394 1 Sepharose with a shorter linker. We have also attempted to characterize the binding of ICAM-1 to control-DAH Sepharose by comparing its binding with other resins to determine the specificity of the 1-amino-6-acetoamidohexane Sepharose 4B/ICAM-1 interaction.
Hyaluronan is a negatively charged glycosaminoglycan that occurs in connective tissue and has a wide range of mechanical and cell biological functions. The purpose of this study was to test the ability of various column chromatography resins to bind both hyaluronan and proteins from liver, the major organ of hyaluronan clearance from the blood. The methods used include chromatography of hyaluronan and rat liver endothelial cell extracts on columns of substituted Sepharose matrices, and a simple tube binding assay to test the affinity of detergent-solubilized rat liver intercellular adhesion molecule-1 for these same resins. Using these methods we have found that hyaluronan binds to a hexamethylene chain with either a terminal primary amine or a terminal acetoamido group. This interaction is not simply of a hydrophobic nature, as hyaluronan does not bind the hydrophobic resins hexyl- or octyl-Sepharose. We have also found that intercellular adhesion molecule-1 binds best to resins containing a hexamethylene chain. Finally, we have determined that resins substituted with hyaluronan linked via an ethylene chain can be used to specifically purify hyaluronan binding proteins from rat liver endothelial cells.
In conclusion, this study demonstrates the efficacy of a number of chromatographic resins for binding hyaluronan, hyaluronan binding proteins of approximately 200 and 400 kDa on rat liver endothelial cells, and rat liver intercellular adhesion molecule-1.
Figure 1 relates to diagrammatic representation of the various Sepharose resins, HA-ASD and HMBA.
Figure 2 relates to binding of rat liver intercellular adhesion molecule-1 to 1-amino-6-acetoamidohexane and its displacement by hyaluronan.

Figure 3 relates to binding of rat liver intercellular adhesion molecule-1 to 1-amino-6-acetoamidohexane Sepharose 4B and its displacement by hyaluronan.
Figure 4 relates to binding of rat liver intercellular adhesion molecule-1 to 1-amino-6-acetoamidohexane Sepharose 4B and its displacement by hyaluronan.
Figure 5 relates to binding of rat liver intercellular adhesion molecule-1 to 1-amino-6-acetoamidohexane Sepharose 4B and its displacement by hyaluronan.
Figure 6 relates to binding of rat liver intercellular adhesion molecule-1 to 1-amino-6-acetoamidohexane Sepharose 4B and its displacement by hyaluronan.
Figure 7 relates to chromatography of HA on control-EDA-, P-HA-DAH-and P-control-DAH-Sepharose.
In this study we show that HA, typically considered a hydrophilic molecule, and intercellular adhesion molecule-1 (ICAM-1), a glycoprotein predominantly expressed on vascular endothelium, that mediates neutrophil and macrophage extravasation in inflammatory states (Dustin et al., 1986;
Springer, 1990), have an affinity for both HA-DAH-Sepharose, a resin with HA
attached via the aminohexane (AH) group, and "control-" DAH-Sepharose, a resin containing a blocked aminohexane group but no ligand. Furthermore, we show that neither HA nor ICAM-1 bind HA attached to Sepharose with a shorter linker, or the equivalent control resin. We have also tested a range of other resins for their ability to bind both these and other biomolecules.

MATERIALS AND METHODS
Chemicals Collagenase (Grade V), hyaluronidase (bovine testes type I), leupeptin, pepstatin A, PMSF, benzamidine, N,N'-hexamethylene bis-acetamide (HMBA), poly-L-glutamic acid, ethylene diamine and Triton X-100 (molecular biology grade) were obtained from Sigma Chemical Co., St. Louis, USA. Aprotinin was obtained from Bayer, Leverkusen, Germany. l25I was obtained from Nordion Inc., Canada. Broad-range pre-stained molecular weight standards were obtained from Bio-Rad Laboratories, Hercules, USA. 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) was obtained from Fluka Chemika-BioChemika, Buchs, Switzerland. Cyanogen bromide-activated Sepharose 4B, EAH Sepharose 4B and phenyl Sepharose were obtained from Pharmacia Biotech, Uppsala, Sweden. AH
5 Sepharose 4B was a gift of Professor Jan-Christer Jansson, Pharmacia BioProcess Technology, Uppsala, Sweden. HA-DAH-Sepharose, produced using Pharmacia's AH Sepharose 4B as starting material for the coupling of HA
(Tengblad, 1979) was a gift of the late Dr. Anders Tengblad, Pharmacia, Uppsala,Sweden, and is referred to in the text as P-HA-DAH-Sepharose. The HA
10 Radiometric assay was purchased from Pharmacia Diagnostics, Uppsala, Sweden.
Hyaluronan (approximately 900kDa) was a gift from Ove Wik, Pharmacia, Uppsala, Sweden and Healon (approximately 3 x 103 kDa hyaluronan) was a gift of Pharmacia, Uppsala, Sweden. Heparin was a gift of Dr. Marco Maccarana, Department of Medical and Physiological Chemistry, Uppsala, Sweden.
Preparation of HA for coupling to Sepharose and for elution of bound proteins.
HA was prepared as described previously (Forsberg and Gustafson, 1991).
Briefly, HA was digested with bovine testes hyaluronidase and the resulting oligosaccharides chromatographed on a calibrated S-300 size exclusion column.
20 HA fractions corresponding to 25-40 kDa were pooled and dialyzed against ammonium bicarbonate before being lyophilized to dryness. The resulting lyophilisate was dissolved either in water for coupling to aminoethyl-oraminohexyl-Sepharose or in phosphate buffered saline (PBS) (pH 7.4) (137mM
NaCl, 2.7mM KCl, 6.5mM Na2HPO4 and 1.5 mM KH2PO4) for elution studies.
Preparation of HA-ethylenediamine Sepharose 4B and 1-amino-2-acetoamidoethane Sepharose 4B.
HA-ethylenediamine (HA-EDA) Sepharose 4B (Figure lb) was prepared as follows. Aminoethyl (AE) Sepharose (Figure la) was prepared according to Cuatrecasas and Anfinsen (Cuatrecasas and Anfinsen, 1971). Washed cyanogen bromide activated-Sepharose 4B was suspended in an equal volume of water containing 2 mmoles ethylene diamine (pH 10) per ml of Sepharose. The gel slurry was mixed end-over-end at 4~C for 16 hours and then washed on a sintered glass funnel with two cycles each of lM NaCl, 100mM NaHCO3, pH 10.0 followed by lM NaCl, pH 4.5 (70ml buffer per ml Sepharose). The aminoethyl-Sepharose was washed further with 0.5M NaCl, pH 4.5 and finally with water, pH
4.5 and then mixed with an equal volume of water at pH 4.5 containing 9.5 mg/ml hyaluronan (mean Mr=36000). EDC was then added to a final concentration of 2.3 mM (corresponding to a HA disaccharide:EDC ratio of 10:1) and the slurry mixed end-over-end at room temperature for 24 hours. The pH
was maintained at 4.5-5.8 during this time by the addition of 0.1M hydrochloric acid. The resin was washed as before and then mixed with an equal volume of

3.9M NaCH3COO, pH 4.5 before the addition of EDC to a final concentration of 100mM, and the slurry mixed end-over-end at room temperature for 24 hours in order to block the remaining unsubstituted amino groups on the gel. The resin was washed as before and stored in an equal volume of PBS containing 0.02%
(w/v) NaN3, to form a 50% (v/v) slurry for dispensing of the resin.

The control resin 1-amino-2-acetoamidoethane (control-EDA) Sepharose 4B (Figure 1c) was prepared in parallel, but with the elimination of the HA
oligosaccharide/2.3mM EDC coupling step.

Preparation of HA-1,6 diaminohexane Sepharose 4B and 1-amino-6-acetoamidohexane Sepharose 4B.
HA-1,6 diaminohexane (HA-DAH) Sepharose 4B (Figure ld) and 1-amino-6-acetoamidohexane (control-DAH) Sepharose 4B (Figure le) were prepared in parallel with the previous two resins, but with the substitution of the longer linker 1,6-diaminohexane for ethylene diamine in the first step.

-14- 21939~1 Two further l-amino-6-acetoamidohexane Sepharose 4B resins were prepared using either AH Sepharose 4B, a resin produced by Pharmacia BioProcess Technology from the coupling of 1,6-diaminohexane to CNBr-activated Sepharose 4B, or EAH Sepharose 4B (Figure lf), a resin produced by Pharmacia Biotech from the attachment of the same linker to Sepharose 4B via an epoxy coupling method. These control resins were prepared as was the control-DAH-Sepharose with the elimination of the 1,6-diaminohexane coupling step and are referred to in the text as P-control-DAH-Sepharose and control-EDAH-Sepharose (Figure lg), respectively.

Preparation of hydrophobic resins of various chain length.
Ethylamine, n-propylamine and n-butylamine in water, pH 10, and n-pentylamine, n-hexylamine, n-octylamine, n-decylamine and n-dodecylamine in 50% (v/v) dioxane, pH 10, were coupled to CNBr-activated Sepharose 4B at 4 mmoles per ml resin, under the same conditions as for the ethylene diamine couplmg.

Assay of resins for the presence of coupled HA.
Aliquots of 100111 of 50% (v/v) slurries of the various Sepharose resins were each equilibrated in 0.15M NaCl, O.lM NaCH3COO, pH 5.0 and then incubated with 40 units of bovine testes hyaluronidase in 550,u1 of the same buffer, end-over-end for two hours at 37~C. The resins were allowed to stand overnight at 4~C before being remixed and centrifuged. Their respective supernatants (500~1) were then assayed for the released HA
oligosaccharide/glucuronic acid content (Bitter and Muir, 1962). The HA-EDA-and the HA-DAH- Sepharose used in this report were substituted with 0.22 and O.llmg HA/ml wet gel, respectively. The HA-DAH-Sepharose provided by Dr.

Anders Tengblad, denoted P-HA-DAH-Sepharose, was substituted with 0.7mg HA/ml wet gel. There was no detectable HA on any of the control resins.

Chromatography of HA on various HA- and control-resins.
Glass columns of 7.5mm diameter were packed with 4ml each of hexyl-, octyl-, AE-, HA-EDA-, control-EDA-, control-DAH-, P-HA-DAH-, P-control-DAH-, EAH- and control-EDAH-Sepharose, and equilibrated with PBS containing 0.1%
(v/v) Triton X-100. HA oligosaccharides (0.95mg in 0.1ml H2O) were pipetted directly to the gel bed of each column and then run into the resin surface. A
200,u1 aliquot of equilibration buffer was then similarly applied and run into each resin before a 400~11 cushion of the same buffer was pipetted onto the gel bed.
The columns were then connected to pre-column peristaltic pumps and washed with four volumes of the same buffer under continuous flow at approximately 2ml/h. Fractions were collected at 15 minute intervals and assayed for the presence of HA using the HA Radiometric assay kit from Pharmacia Diagnostics according to the manufacturers instructions. All steps were performed at room temperature.

Isolation of rat liver ICAM-1 Rat liver ICAM-1 was isolated as previously described (McCourt et al., 1994). Briefly, Triton X-100 solubilized protein from (routinely) 20 homogenizedrat livers was sequentially chromatographed on wheat germ agglutinin-Sepharose, Reactive Yellow 86-agarose, Reactive Blue 4-agarose, Concanavalin A-Sepharose, HA-1,6 aminohexane-Sepharose and finally again on Concanavalin A-Sepharose as a concentration step.

Isolation of rat liver endothelial cells.
Single cell suspensions were prepared from the livers of male Sprague-Dawley rats by collagenase perfusion according to Obrink (Obrink, 1982). LEC

were isolated following Percoll gradient centrifugation and selective adherence according to Smedsr0d and Pertoft (Smedsr0d and Pertoft, 1985) and either solubilized directly in PBS containing 1% (v/v) Triton X-100 and protease inhibitors (lOmM benzamidine, 50 KIE/ml Aprotinin, 0.2mM PMSF, 1~g/ml leupeptin and 1~1g/ml pepstatin A) with or without 0.5 mM EDTA, or cultured overnight at 37~C in RPMI 1640 medium on fibronectin coated plates for surface labeling experiments (see below).

Surface labeling of rat liver endothelial cells.
Overnight cultures of approximately 10 x 106 LEC on 60cm2 plates were surface labeled with l25I using the lactoperoxidase method (Hubbard and Cohn, 1972) as previously described (Forsberg and Gustafson, 1991). The cells were washed six times with PBS and then solubilized in PBX/1% (v/v) Triton X-100 (lml) containing protease inhibitors (lOmM benzamidine, 50 KIE/ml Aprotinin, 0.2mM PMSF, 1~1g/ml leupeptin and 1~g/ml pepstatin A) with or without 0.5mM EDTA.

Whole rat liver and rat LEC ICAM-1/Sepharose matrix binding assays Aliquots of 20111 (for whole rat liver ICAM-1 binding studies) or 40,u1 (for rat LEC extract binding studies) of 50% (v/v) slurries of various Sepharose resins were each equilibrated in PBS containing 0.1% (v/v) Triton X-100 and 10mM
Benzamidine (PBS-TB), centrifuged briefly and all supernatant decanted. Whole rat liver ICAM-1 (approx. 0.02~g as determined from limiting dilutions of protein analyzed with SDS-PAGE (Laemmli, 1970) and silver staining (Morrissey, 1981)) in PBS-TB (10111) or rat liver endothelial cell extracts (approx. 60,ug total protein, determined using the Pierce BCATM Protein Assay, with BSA as standard) in PBS-TB (200~11) were applied to each gel and mixed every 10 minutesfor 1 hour at room temperature. The various agents (HA (5mg/ml), heparin (5mg/ml) poly-L-glutamic acid (5mg/ml), HMBA (25mg/ml), NaCl (0.75 and -17- 2~ 93q41 1.5M) and EDTA (0.5 and 5mM) - final concentrations in PBS-TB indicated) were also added separately in some experiments to test their effect on the binding ofICAM-1 to control-DAH-Sepharose and P-control-DAH-Sepharose. The supernatants were decanted and retained for analysis and the resins washed threetimes with PBS-TB (lml). The material that bound to the resins was released by mixing the resins with SDS-PAGE sample buffer containing 4% (w/v) SDS, 29%
(w/v) sucrose, 0.008% (w/v) bromophenol blue and 0.08M Tris/HCl, pH 8.8 (20,u1) and boiling the mixture for 3 minutes. Bound and non-bound material was then analyzed by SDS-PAGE and immunoblotting (Bjerrum and Schafer-Nielsen, 1986) with rabbit anti-HARLEC polyclonal antiserum (Forsberg and Gustafson, 1991) that recognizes rat ICAM-1 (McCourt et al., 1994) diluted 1:2000.

Elution of proteins from HA-EDA-, control-EDA-, HA-DAH- and control-DAH-Sepharose with HA oligosaccharides.
Columns (17mm diameter) were packed with three ml each of HA-DAH-, control-DAH-, HA-EDA- and control-EDA-Sepharose and equilibrated with PBS
containing 0.1% (v/v) Triton X-100 plus protease inhibitors (lOmM benzamidine, 50 KIE/ml Aprotinin, 0.2mM PMSF, 1~1g/ml leupeptin and 1,ug/ml pepstatin A) with or without 0.5mM EDTA. To determine which cell surface proteins bound to the above resins, Triton X-100 extract (corresponding to approximately 3 x 106 cultured LEC) of 125I-surface labeled LEC were diluted to 0.1% (v/v) Triton X-100 with PBS (containing the above protease inhibitors with or without 0.5mM
EDTA) (3.0ml) and applied to the columns at 0.6ml every 5 minutes. The columns were washed with equilibration buffer (15ml) and then a 500,u1 aliquot of 10mg/ml HA oligosaccharides was applied. The columns were washed thereafter with equilibration buffer (500,u1 every 10 minutes) and fractions collected. All chromatographic steps were performed at 6~C. Fractions containingmost radioactivity were analyzed by SDS-PAGE and autoradiography or immunoblotting with the rabbit anti-HARLEC polyclonal antibody.

-RESULTS
Binding of HA to various resins.
To determine which matrices had some affinity for HA, HA
5 oligosaccharides were chromatographed on various resins, in the presence of 0.1% Triton X-100. Of the HA applied (0.95mg) to the resins, 100% was recovered from the hexyl- and octyl-Sepharose and from HA- and control-EDA-Sepharose (after elution with 4 column volumes of buffer), 61% was recovered from AE-Sepharose while only 23%, 22%, 16% and 7% of that applied was recovered from 10 the control-DAH-, P-HA-DAH-, control-EDAH and P-control-DAH-Sepharose, respectively. Only 16% of the applied HA was similarly recovered from EAH-Sepharose. Shown in Figure 2 are chromatograms of HA eluted from control-EDA-, AE-, P-HA-DAH- and P-control-DAH-Sepharose. Trailing of HA was noted with all resins, with the exception of hexyl- and octyl-Sepharose, which 15 eluted as sharp peaks.

Binding of ICAM-1 to HA-DAH-, Control-DAH-, HA-EDA- and Control-EDA-Sepharose.
To determine the affinity of ICAM-1 for various resins, a simple 20 tube/resin assay was developed. Both ICAM-1 (purified from whole liver) and LEC extracts were used as starting material for binding assays. Immunoblots of bound protein eluted from the resins by boiling in SDS show that more ICAM-1 from whole liver (Figure 3A) binds to P-control-DAH-Sepharose (lane 6) than to P-HA-DAH-Sepharose (lane 5), though it should be noted that the P-HA-DAH-25 Sepharose used in this experiment was synthesized in the late 1980's. However,this same pattern is also apparent with more ICAM-1 binding to control-DAH-Sepharose (Figure 3a, lane 4) than HA-DAH-Sepharose (Figure 3a, lane 3), although these resins bind less ICAM-1 than the previous two. There is no binding of whole liver ICAM-1 to either control-EDA-Sepharose (Figure 3a, lane 2) or HA-EDA-Sepharose (Figure 3a, lane 1). The non-binding fractions in the supernatants were also analyzed by immunoblotting. Figure 3(a) shows that the most ICAM-1 remains in the supernatants of control-EDA-Sepharose (lane 7) and HA-EDA-Sepharose (lane 8), less remains (in descending order) in those of HA-5 DAH-Sepharose (lane 9), control-DAH-Sepharose (lane 10) and P-HA-DAH-Sepharose (lane 11), and virtually none remains in that of P-control-Sepharose (lane 12). The band in Figure 3(a) lane 13 represents the amount of ICAM-1 applied to the resins in this experiment. The effect of EDTA on the binding of whole liver ICAM-1 to P-control-DAH Sepharose was also tested. Figure 3(b) 10 shows that the amount of ICAM-1 binding in the presence of 5mM EDTA (lane 2) is reduced relative to that in the absence of EDTA (lane 1). In experiments using LEC extracts it was found that similar amounts of LEC ICAM-1 bound to both HA-DAH- and control-DAH-Sepharose but to neither HA-EDA- nor control-EDA-Sepharose (results not shown; see also Figure 6). The amount of LEC extract 15 ICAM-1 binding to HA-DAH- and control-DAH-Sepharose relative to that applied was rather less than if purified whole liver ICAM-1 was used (results not shown; see also Figure 5b). It should be noted that when the non-bound- and SDS-eluted-fractions were decanted for immunoblotting, there remained material in the liquid-phase of the Sepharose gel which was not assayed. It is 20 therefore that the amount of bound plus unbound material appears to be less than that applied to the Sepharose resins.

Binding of ICAM-1 to other matrices To investigate if the binding of ICAM-1 to control-DAH-Sepharose was of 25 a hydrophobic nature, as suggested by the requirement of the extra four methylene groups in the DAH linker, we tested a range of hydrophobic affinity resins with increasing aliphatic chain length for their ability to bind ICAM-1.
ICAM-1 purified from whole rat liver was incubated with ethyl-, propyl-, butyl-,pentyl-, phenyl-, hexyl-, octyl-, decyl- and dodecyl-Sepharose, as well as control-21 9394 i DAH-Sepharose, and bound protein eluted with SDS and analyzed with immunoblotting as before. There was no detectable ICAM-1 binding to the ethyl-, propyl- and phenyl-Sepharose resins (Figure 4, lanes 1, 2 and 5, respectively), some binding to the butyl-, dodecyl-, decyl-, octyl- and pentyl-Sepharose resins (increasing in that order) (Figure 4, lanes 3, 9, 8, 7 and 4, respectively) and more binding to the hexyl- and control-DAH-Sepharose resins (roughly equal) (Figure 4, lanes 6 and 10, respectively). However, P-control-DAH-Sepharose bound more ICAM-1 than control-DAH-Sepharose (Figure 3a, compare lanes 4 and 6). The band in Figure 4 (lane 11) represents the amount of ICAM-1 applied to the resins. Non-substituted cross-linked Sepharose 4B and Tris-blocked cyanogen bromide Sepharose did not bind ICAM-1 (data not shown).

Effect of HA, heparin, poly-L-glutamic acid, HMBA and NaCl on the binding of ICAM-1 to P-control-DAH-Sepharose To investigate which agents could inhibit the ICAM-1/ 1-amino-6-acetoamidohexane Sepharose 4B interaction, purified whole rat liver ICAM-1 or LEC extracts, in the presence of various agents, were incubated with aliquots of P-control-DAH-Sepharose. Bound protein released by SDS was analyzed as before.
The band in Figure 5a (lane 1) is the amount of ICAM-1 binding to P-control-DAH-Sepharose in the absence of any additives. HA at a concentration of 5mg/ml significantly inhibits the binding of ICAM-1 from whole liver (Figure 5a,lane 2) to P-control-DAH-Sepharose. Heparin (5mg/ml) apparently had no effect on the binding of whole liver ICAM-1 to P-control-DAH-Sepharose, though two extra immunoreactive bands at approximately 60 kDa were also evident (Figure 5a, lane 3). The same concentration of heparin actually caused significantly greater amounts of immunoreactive material from LEC extracts to bind control-DAH-Sepharose (Figure 5b, lane 2), than that which bound to the same resin in the absence of heparin (Figure 5b, lane 1). The broad band in Figure 5(b) lane 2extended from 85 kDa to approximately 55 kDa. Poly-L-glutamic acid (5mg/ml), -21- 2 1 939~ 1 while not causing any significant reduction in whole liver ICAM-1 binding, did result in a distorted ICAM-1 band migrating at a slightly higher Mr (Figure 5a, lane 4). HMBA, even at a concentration of 25mg/ml, had no effect on the binding of ICAM-1 from whole liver to P-control-DAH-Sepharose (Figure 5a, lane 5). NaCl at concentrations of 0.75 and 1.5M (Figure 5a, lanes 6 and 7, respectively) reduced the binding to some extent, but not to the same degree as HA (Figure 5a, lane 2). The bands in Figure 5(a) lane 8 and Figure 5(b) lane 3 represent the amount of ICAM-1 applied in the respective experiments.

Elution of ICAM-1 and other proteins from control-DAH-Sepharose and other resins.
To demonstrate that ICAM-1 and other proteins could be eluted from control-DAH-Sepharose, mini columns of the same resins, as well as HA-DAH-, control-EDA- and HA-EDA-Sepharose were used to affinity purify material from Triton X-100 extracts of 125I surface labeled LEC in the presence and absence of0.5mM EDTA. Immunoblotting revealed that HA elutes comparable amounts of ICAM-1 from HA-DAH and control-DAH-Sepharose in the absence of EDTA
(Figure 6, lanes 3 and 4, respectively) but autoradiography of the same materialrevealed that many other protein bands, besides those in the 80-90 kDa region, were eluted also from the same resins (Figure 7a, lanes 3 and 4, respectively).
Similarly, in the presence of EDTA several bands were eluted from both HA-DAH and control-DAH-Sepharose (Figure 7b, lanes 3 and 4, respectively). No detectable amount of ICAM-1 was released by HA from HA-EDA- and control-EDA-Sepharose in the absence of EDTA as analyzed by immunoblotting (Figure 6, lanes 1 and 2, respectively). However, proteins which appeared to bind more specifically to HA were observed in the eluate from HA-EDA-Sepharose. From this resin HA eluted 400, 200 and 84 kDa bands in the absence of EDTA (Figure 7a, lane 1), and similar bands in the presence of 0.5mM EDTA, though the 84 kDa band was markedly reduced in intensity (Figure 7b, lane 1) and was not apparent -22- ~ l 9394 1 in all experiments. From control-EDA-Sepharose HA eluted 220 and 84 kDa bands in the absence of EDTA (Figure 7a, lane 2), though the 220 kDa band was not apparent in all experiments, and only small amounts of the 84 kDa component in the presence of EDTA (Figure 7b, lane 2). It should be noted that 5 the estimation of the molecular weight of the 400 kDa band is approximate, as we did not have standards larger than 207 kDa.

DISCUSSION
HA, a polyanionic polysaccharide, is typically considered a hydrophilic 10 molecule. However, another feature of HA is the existence of large hydrophobic patches (of eight abutting CH groups) extending over three disaccharide units, repeated on alternate sides of the molecule throughout its length (Scott, 1989). In aqueous solution there is also extensive H-bonding within the HA molecule, via H2O bridges, between the acetoamido groups on N-acetylglucosamine residues 15 and the carboxyl groups on glucuronic acid residues. The internal H-bonding causes considerable local stiffness in the chain (Laurent, 1970; Scott, 1989) and, together with interactions between the hydrophobic patches, also allows HA to form networks with itself and possibly other components of connective tissue (Laurent, 1970; Scott, 1989; Scott et al., 1991). These characteristics of HA, together 20 with its high molecular weight, may in part explain the unique visco-elastic properties of HA.
In this study we have tested a range of substituted Sepharose resins for their ability to bind HA in the presence of Triton X-100 (see figure 2 and results).
We have found that HA has some affinity for resins containing a hexamethylene 25 chain with a terminal amine (Figure lf) or a terminal acetoamido group (Figure le and g), less affinity for a resin containing an ethylene chain with a terminal amine (Figure la), and no affinity at all for a resin with an ethylene chain and a terminal acetoamido group (Figure 1c). This showed that there was a requirement for a chain longer than two methyl groups for HA binding to the 2193~41 acetoamido resins, suggesting a hydrophobic interaction. However, as there was no HA binding to either hexyl- or octyl-Sepharose, the binding was not simply ofa hydrophobic nature, but also required the presence of a terminal acetoamido group. Thus, the six methyl groups and the terminal acetoamido group may act in concert to bind the hydrophobic patches and carboxyl groups on HA. It is therefore possible that 1-amino-6-acetoamidohexane group on resins depicted in Figure le and lg represents a novel HA binding moiety, which may also provide an alternative means for the purification or concentration of HA. Further studies to find the optimal aliphatic chain length (in this moiety) for HA binding are warranted. The isourea substituent, which results from the reaction of the cyanate ester on CNBr activated-Sepharose with amino compounds, is positively charged at neutral pH and may further act to stabilize the binding of the negatively charged HA to control-DAH-Sepharose (Figure le) though this is not an essential requirement for HA binding. The resins containing a terminal amine at the end of an aliphatic chain, namely AE-Sepharose (Figure la) and EAH-Sepharose (Figure lf), also bound HA. In the case of AE-Sepharose, this is most likely an ionic interaction between the positively charged (at neutral pH) primary amine and the negatively charged HA. However, the extra four methyl groups on EAH-Sepharose seemed to greatly enhance the binding of HA.
It has previously been reported that glycosaminoglycans can be chromatographed on hydrophobic resins such as phenyl- and octyl- Sepharose in the presence of 0.01M hydrochloric acid and high concentrations (4.0 - 2.0M) of ammonium sulphate (Nagasawa and Ogama, 1983; Uchiyama et al., 1985). These high salt concentrations were necessary for the binding of polysaccharides to the resins, which were then eluted by decreasing salt gradients. As the behaviour resembled a hydrophobic interaction between polysaccharides and resins the process was originally considered to be hydrophobic interaction chromatography (Nagasawa and Ogama, 1983). However, subsequent analysis showed that the retention of the polysaccharides on the columns was related to their solubility in ammonium sulphate (Uchiyama et al., 1985) and the authors concluded that the fractionation depended on the ability of the polysaccharides to precipitate on the gel rather than hydrophobic interactions. (See United States Patent 4,421,650 (Nagasawa et al. See also Uchiyama, H., Okouchi, K., and Nagasawa, K. [1985]
5 Chromatography.) We have also tested some of the above resins for their ability to bind partially purified proteins from rat liver and LEC. These studies were necessaryin light of our previous findings that a 90-lOOkDa protein bound to and could beeluted (with free HA) from HA-DAH-Sepharose (Forsberg and Gustafson, 1991;
10 McCourt et al., 1994). Early control experiments indicated a lower binding to a control-DAH-Sepharose than to a HA-DAH-Sepharose, of radiolabeled material that had been affinity purified on the latter resin (Forsberg and Gustafson, 1991).
After the identification of the protein as rat ICAM-1 (McCourt et al., 1994), studies were initiated to identify potential HA binding sites in fragments of ICAM-1. It15 was then found that considerable amounts of ICAM-1 bound to both HA- and control-resins (Figure 3). This led to a further investigation of the nature of the affinity chromatographic process.
During the course of these studies, we have found that the most effective resin for the purification of HA binding proteins from rat LEC, with the least 20 non-specific binding, was HA attached to Sepha~ose via a short ethylelle rlj;~min~ base linke~ (Figure lb). We foulld thal ICAM~ not bind this resi~ nor to the equivalent ~ontrol re~in (Figure 3). The fact ~hat these resins ~vere boiled i~
the p~esence of such a potent den~g ~ge~ as SI~S~ ~d tha~ ~he majo~i~ o ~ ICA~ ined in the s~ ~nl ~Fig~e 3a, lanes 7 and 8) ma:~es i~ vnli~ly that a~y ICA~-l was ~c)und t~ A-andcon~c~1-EI)A-Seph~ose. H~wever, that solu~ 7e i ICAM-1 does not bind HA-Er)A-~ph~rose does not exclude ~e possibili~ that ICAM-1, ullder physinlo~ l cond~ n-e~ has some af~nity fo~ ~.
It as bee~ ~;ho~ ~at s~ bili7~rion wi :h cletergent ca~ c~ange ~e affiniy an i sperifici~ of a HA~ell memh~ane ~eptor (Unde~hill et a1., 1~8~;). We have now i~enti~e~ two n~w bands of r~ tely) ~ao alld 2001~ that s~ri{;r~lly bind to and elute ~ith H~ m HA-E~A-serh~rose IFigure 7).

J ~hy ~1l HA-S~rh~rose h~s been widely used.
Althou~h Inost au~ors do ~ot specify t~e na~ of the space~ ~m co~lp!inE ~3A to th.e res~n, HA-DAH-Seph~rose 1~; probably bee~ ~he most c-~nrl~nly u~d gel. It is ther~l~e of g~ral interest to analyse t~ ity process in questlo~. In this study we s~ow firstly thAt sev~a~ LEC sur~ace proteins, includirlg ICAM-l, can be elut~d wit~ llgos~ccha~ides ~rom both HA- cmd contr~l-~AEI-~eph~rose (~igures 6 ~d 7), and se~ondly that llA actually has an affinity foI P-EA- a~d P~ontroI-DAH~Seph~ose ~Figure 2) ~nd othe~ similar resi~s used iI- this study. Ther~fol~ it is n~t I~ossib to use the ~riteri~ of affiniy elu~on of ~l~t~S ~uth H.4 from either HA- or con~ro~ seph~ros~ as a means ~o defin~ HA
receptors, as all of t;hese protei~ ~ands would then r~p.~ese~t potenlial c~n~ te LEC receptors for HA~ or ~ ted proteins. I~

2~

~y case, I:ne ~l~ffon o~ t~ese proteins m~y not be a speciflc affi~
ph~nc~ on, b~t instead s~ply a dis~l~r~ment phen~ mçnon whereby EIP~ competes ~n~ ~ese proteins for ~e same lig~nd namely l-.~nin~6-aceto~midQ~ e on con~ol-D~H-seph~rose 4B
(Figure ld). This ligancl s~oul~ o e~as~ on the P-HA- and HA-Sepha~ose as a I-~sult of the blo~ of unsubsti~utecl ami~o ps on t~e ~esiIl ~th acet;3te. ~t is irlLeles~g ~hat of tb~ee pol~o~;, n~y HA, heparin an~ poly-L-gl~l?/mic acid~ only HA
could inh~bit ~CAM l bin~ o P-con~ol-SPph~rose (Figllre S) S~ C~in~ t neither hepa}in nor poly-L~lut~mic acid h~s as muc~ affi~i~ for ~is resin ~s does HA. It would be of illterest ~ves~gat~! whed~er other polysa~char~des, such as der~ t~n slllph~te, chond~oitin sulphate or k~n sulphate, h~ve any affi~ity fol ~is ~
~n ~his stu~iy we have at~ pte~l to iden~fy the moie~ t~ ~vhich ds on con~ol-~AH-Sepharose ( 1-~o-6-~et~mi~ ne S~ rose 4~). We have show~ that P~o~trol-I~AH-~epharose m~de using Pha~ia's AlI-Sephar~se 4B bi~ds ~e mt)st ICAM-1 of ~11 resi~s tested. Th~ ~e con~ AH-seph~ose~
made ~h 1,6~i~min~ xane and CNBr-~c~vat~d-Sepha~ose, did not bind K'A~ s efrecli~re~y is prob~bly due to dlat we d~d not have op~lal condi~ions for t~e coupli~g of ~ c the manuf~cturer would h~Ye ~een lik~ly to use. This is also a p~s~ihle n~1inl~ fc~r the reduced amou~t of HA coupled to our HA-EDA-and HA-D~-Sep~ose (0.22 ~nd 0.11 mg HA/Inl wet gel, ~espec~vely) rela~ve to ~t c~lple~1 to P-HA-I~AH-Sepba~ose (0.7 mg HA/ml wet ~el), al~ough the use of lower Ersc (cal L,aiiil lide) co.l~n~rations ~ our coupling mixtures may also be a cont~ibu~g 2 1 9~4 1 facto~. In s~ies ~th oeher resi~s m~de using C~Br~c~vat~d-Sepharose. it ~as found ~at he~l-S~ph~rose ar~d con~ol~
Sepharose bound c~mp~r~ble amounts of ICAM-1 (Figure 4), ~hough con~ol-~-Sepharose bou~ affler less th~ P~on~rol-I;~
Sepharose (Fl~ure 3). That l:he oc~l-~ decyl- a~d dodecyl-Sepharose r~s bound less ICAM-1 may be dlle. tO I'~dUCed subs~ c~ ~y these ligant~s ou~ng to ~hPi wer solubility in aqueo~s st~lu~ons, eYe~ ~ the presence of SO% (v/v) dioxane. The ~mpo~ta~ce of the SiY ca~ s on ~he control resin, wi~ ~e te~
amide g~Ollp $uggested that ~e ~n~neopla~c agent HMBA (Marks et al., lg~ (Figure lf~ Inight ha~re been a liga~ld for ICA~-l.
Hc~wever, lhe fln-31~ that eve~ quite bigl~ concen~l:ions of H~A
(Z5m~ ould not hincler I~ l hi~-~l;ng to the coll~rol-Sepharose (Pi~ure ~) indic~ h~t this is not ~e case. The ~er~clioll of IC4M-l wi~ch ~he same re~ vvas inhibited somewhat with high ~:on~en~tio~s of NaCl, ~ough not to the same degree as wi~ch HA (Figure 5) which s~gge$~s ~at ~he ~in~lin~ is not pu~ely of a hydnop~ic D~ture. T~us~ Y~n~ at~a~hed by the cya~
bromide li;~kage ~o SP~h~ose ~ria a secondary amine may represent a new l~a1ld for rat ICA~ i may provide a nov~ me~nc of ptl~g l~

Hepa~ aE~pea~ed to e~ e ~e ~inc~ of i~ oreact~ve Pri~ om LEC, to P-co~t:rol-~AH-Seph~r~se, r.esuldng ~ a broad dislorted ~ d of a lower avera~e MI~ n that of ICAM-l (Figure 5b). Some i.~ ore~ve n~terial of low~ Mr was al$o elute~
from the s.~me re~ ft~ cub~rion with whole liver ICAM-l ~d heparin (~ Sa). This may sunply be a case of pIotei~
precipi~ n~ du~ing the chIom~to~raplJic step, by ~is sulIlh~

bi~bly negat;lvely charged polysacc~de, whic~ can interact with m~y cat~ nic biological molecules. This would e}~lain why rela~vely ~nole i~nn~unoreac~ve nL~te~l was obt~inetl whe~ LEC
~x~acts w~re applied to ~e ~e~i~ in the p~esen~e of hep~.

The range of prot:ein b~ s in Figllre 7 were nc~ see~ in tlle initial study of Forsberg and Gustafson (1~91). This w~s possibly ~lle to ~e fact th;~t ~e protease i~lhibitor coc~ ~ed in ~t s~udy ;nrl~l~erl only tch.~ seri~e p~otease inhikit~rs P~ISF a~d Aproti~, which wer~ insufflcellt to preve~t even the degrada~n of ICAM-1 in L~ge sc;~le p~fi- :~tinn ~ n~p~si in the pr~ s~udy a more exte~sive Ia~ge of protease in~ihi~nr$ was used. Also of po~
importanc~ is that th~ S~h~os~ used ~ that and later studies (Gl~s~s~ and ~o~g, lg91; h~cCour~ et al~ 4) was coupled ~ccording to ~e Tengblad met:hod (Teng~lad, 1~7~) where ~e HA dislcc~ride:E~C mola~ o was 1:5, while in the p~esent s~dy a r~liO of 10:1 was used, which wo~lld ~estllt in lowe~
subs~tu~on of ~:he poly.c~r~h~ride chai~. Howe~e~, the lar~e exces~
of EDC used i~ ~he T~ me~hod did ~ot caLIse ~ ffic~t chetnic~l nlo(iiflc~on of the collpled ~A on P~ AH Seph~ose as it s~ cally ~is HA binclin~ prot~s (Tengblad, 197~) ~nd is sensi~ve ~ y~ n~ p ~igesdon.

Several o~er g~oups ha~e also used HP~-~Pph~rose to puIify H4 l?;inr1in~ pr~teins ~h simil~r mole~ar weights ~s ICAM-1 (Mason et ~., lg~c~; Sa~ et al.~ hough they ha~e not speci~d which linkl~ was llsed to ~ouple ~A to ~e ~sin. LeBaron et al.
(199~ ~onlpared the collpling of HA to bo~ ami~oethyl-~ph~rose and ~AH-S~ o~e (a 1~ atom hyd~ophilic spacer a~n formed by the covale~t li~kage of 1,6 d~ ~PY~n~ to .Se~h~ros~ 4B by a~
epoxy coupling tnt~hod) and found no ~irfer~ce in the coup~ng efficie~cy. It wa~ ~is find;n~ ~at p~~ Jted us to use ~ shorter e~hylene di~mine based linke~ give~ the si~e of ~e HA (3~ kI)a) ~e longe~ linke~ would co~fer no advall~ges ~h regard to stenc hindr~ce. T~ ~e use of a long lillker to couple s~ large n~ is unw~ted on ~ce counts. Firstly, ~he longer the linker, :he g~e~ter the risk of non-specifi~ protei~ bin~1ir.~.
Secondly, :~ cotlples as we~l to bol:h short or long lin~er Sepharose.
Thirdly, give~l the size of the HA ligand, a lon~ linker is not necessary to se~ it auay from ~e ma~ix for it ~ bind prot~ln. The ~n~in~of Underhilletal. (1~85) thatHA~ Sepha~ose ch~ t~ ~phy achieved no puri~cal:ion of C1~ cell su~face ~eceptor f~lr hyaluro~an ~ressed o~ of cells of the imn1urle s~scem as well as eryrh~cytes a~d fib~o~l~ts (Underhill, 1~92)~ ha~e ~n due to ~he ~on-specific binding of other prote~s tc~ ~e 10llg DAH linker.

Yan~ ro~ e~ al. ( 1992) h~ve i~en~fied ~sNo large polypep~ddes (166 and 175 kI~a) on ~he su~ace of rat LEC, ~
affinity to ¢~ coupled to a photo~ffint~ cross li~k~ reagent. They also occasi~nally detectecl species of 86, 66 and 55 kD~ Ihoug~
these bands, whe~ evident, varied in i~nsi~ r~ ive ~o ~he 166~175 k[~a doublet. The authors su~ges~ed tha~ l:hese proteins were degr~ ffon product:s of ~ l~rger polypep~des, or HA
bin-linf~ pr~teins on I E¢ unrela~ed to the endoq~to~ re~eptor.
How~ver, a ~ k~a spe~ies on the s~face o~ ~at LEC has recendy bee~ ident~ed as a p~te~tial c~citlm-depe~de~lt HA hinrling protein us~ng ~e ~ne photo~mnity crass li~in~ r~agent ' 2193941 (Yannariell~Bro~ l~g6). We suggested that the ~6 kDa species was ICAM-l and ~h~ 55 k~a species its de~r~ t;~n product (McCou~t 1~ al.~ l9g4). The i~en~ty of ~hese lo~ molec~ weight spe~ies, h~weve~ i..S to be es~hlishecl. The llse of the ~-aLkylpropi~ mi~e-3~ thio-l~yl-Z -~mjllo~ linkeri~ the eross-linking age~t (Figure lh) may have been the cause of so~e non-spee ding of ~hese same prote~ns during ~e photoafflnity l~,bellitt~ l~roCesS. Tbis link~r ha.~ some simi1~ ies tc~ the l-ami~
6-a~etoamido~e~ne moiety on con~ol-l:~AH-SephaIose (Figure le), namely it~ ch~in leng~ and the two a~ido gro~ps en~o~np~.c.cin~
~he link~ l~egio~. T~t ~ presence of ~ lO~fold excess of hyalu~na~ cc~Uld prevent ~he pho~o~ffin~ belli~g of ~e 86 ~n~
55 kr~a prot~s would be consisterlt with our ~lldin~s tl~t f2ee H,4 can displ~e ICA~I-l, and o~her proteins, from con~rol-r)AH-Sepharose by compe~ng for the linke~. The use of the pho~r~
lab~llin~ n~a~e~t witho~t substi~uted HA would perllaps es~hli.ch the specl~l~ity, if ~y, of the la~elling of tlle surface prot~s on LEC.
The sam~ ~ors have ~foposed that ~e ~ L~rge polypep1:ides and ] 751c~a) may exist as ~ ~ercl~ r of ~ v~ tely ~40 kDa ~Yanni~iell~Brown et al., 1997) and h~re ~so fou~d l~5I-HA
bi~ n~ ac~v~ty at ~00 k~a in deter~e~t solubilised LEC ~nenlbranes frac~ion~t~ h gel filt~on (~a~naIiello-Brown and Weigel, 19~). Thls pa~t~, n~mely a la~ge polyp~p~i~e of 340-40Q k~a a~d two s~ r polypeplide~ dose in si~e ~166 a~ 175 kDa), ha~
some simil~ities to the ~NO p~o~ speQes of ~app~ atpl~) 400 and ~00 kl~ ol~ed from HA-EI}A-Seph~ro~e in this study. Work is now undemuay to isolate SUfflCiellt a~ounts of ~ese p~oteills for sequ~ci~ dbody produc~on.

In s~ y, ou~ ~esults have shown ~at ~e l~
aceto~m~ h~ ne group is a poten~ally novel ~IA hinliing moiety.
We also sb~w tha~ the hindin~ of de~etgent solllbi~ ICAM-l to HA atta~h~!ci to ~epharose via a 1,6~i~mitl~he~ane linkerl and its displ~cemt~nt ~rom ~his resi~ ~ free HA oligo$~ch~r~des, ~ ot be used as a me~s to ri~mon~ate that ICAM-l has an af~i~ity for HA. T~}e bin~ g of bo~ HA and ICA~-l ~o the linker ~nde~ ~his unsuit~ble ~or such s~udies. Howe~er, this same flnclin~
sllggests t~t can~ol-I)AH-Sep~arose may provide aIl alterna~ve m~ns to F~U~ andJor conce~trate ~e~ tw~ ilnportant biolo~ical molecules. Final~y, we have also i~pntifie~l ~o proteins from rat LEC which are p~ l c~n~clicl~t~ for the c~ m independe~t end~cyto~c HA le~el?tol.

ACK~IOU~IE:D~EM13NlS

We a~e gra~eful t~ ~Is. ~ajsa Iilja fo~ t~chnic~l assista~ce, a~d to Ms. An~l~arie Gustafson a~d ~. J~ M~kko for PYr~ nt r.lt liv~ p~lci~nc We ~h~nk ~r. Ri~n~'~ Tom~sini-~h~ncson~ ~r. NiCk ponh~m, ~r. Ro~ert ~oulder and F~fesso, Torvard I~u~ent fo~ their invaluable advice ~ disc~1s~io2~-~ during ~s s~dy. We thallk ~r. St:affan ,~h~n.cs~n and Plof~sso~ L;~urerlt fo~ ~heir CIitiCal review of this manusc~ipt. This work w~
s~ porLe,l by AgT~es and Mac Rudbergs fond, E~l~nd Wessle~s fond, KoIlun~ Gu.staf V:s 80-arsfond and dle Sw~dis~ Mediral Resea~ch Council.

21 9394 ~

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-LBGENDS ro ~

Fl~u~e ~ ])ia~ ~p~ t~t~on of ~evarlo~s Serh~rose resl~ d H~, Shown is ~E-Seph~rose ~a), HA-E~A-Sepl~ose (b), con~ol-EDA-Seph~ose (c)? ~A-I)AH-seph~rose (d), ~on~oI-l~AH-Seph~ose (e), EAH-Sepha~ose ~f), con~ol~ AH-SPph~rose (g), 2(p-azidos~ midc))e~}yl-l,3~ hiop~ n~t~HA (~A-AS~) [34~(hJ
and ~A (~)~

Figure ~ ~Lr ~ ;lt~ r~hyofHAon~onl:rol-l~A~ , P-HA-~AH- ;~d P~on~ol-DAH-SPp~ose.

HA (0.~5 Dlg~ i~ H~O (10û ul) was applied to 4 ml coluInns of con~ol-E~A-, AE-, P-~-D~H- aDd P~ont~ol-I~AH-Seph~rose.
F~actions (app~o~-n~t~ly 0.5 ml~ were collected and ~ysed for the presence Q~ HA. Closed squares ~), open squ~res (~), closed ciIcles (~ d open circles (o) in~ir~te HA (~g/fr~c~on) eluted with PBS/0.1% r.r~iton X-ll~ om con~ol-E~A~, AE~, F'~ DAH- a~d P-con~rol-l:lAH-Se~h~o~e, respe~dv~ly.

Figu~e 3 ]~r~ ob~ots of whole liver ICAM-1 ~uted f~om va~io1ls resins wit~ SDSJ p~obed uith l~he a~tl-EARl~C
~body.

Whole live:r ICAM~ he absence of El}TA was :incubated wi~ the various inclic~ esi~ d ~e ~ou~ on-bound materi~
an~lysed ~ nn~blolting (a). Shown is ~o~d/non-boll~d material fi-om HA-EDA-Seph~ose (lane 1/lane 7), co~ol-Er)A-Se~h~rose (lane ~lane 8), HA-r)AH-Seph~rose (la~e 3/lane g), ~ontlol-~I-Sepharose (l~ne 4/Lane 10), P-HA~ Sepharose (lane 5/lalle 11) ~d P-con~ol~ Seph~ro~e (lane 6/la~e 12).
The balld i~ lane 13 rep~se~ts the materi.al applied to ~e resins in this ex~e~ment. The eff~t of 5mM EDTA on ~indin~ was also tested (b). Shown is eluted whol~ live~ ICAM 1, which w~s bound to P-co~ l-DAH-.~eph~rose ~ t:he ~hsp-nce (lanel) and prese~ce (lane 2~ oi E~TA.

P~gure 4 ~ u~lots of whole live~ ICAM-1 eluted from ~a~io~s h'r~op~b~c re~i~s ~ Sl:~S, ~ ~e HARIEC ~nslh~y.

S~own is an immllnoblo~ of w~ole lhJer ICAM~ the absence of E~TA) t~t was inc1lb~ wi~ the ~n~ic~ed r~iins ~d the bound m~te~ .ut~i Wit}l SDS ~rom e~yl-~ph~rose (lane 1), propyl-Sepharose ~lane ~), b~ Sepharose (lane 3)~ pent~ epharose (Lane 4), phenyl-Sepharose (L~ne S), ~exyl-Seph~ose (lane ~, octyl-Sepharose (lane 7), de~yl-~epha~ose (Lane 8), dodecyl-Seph~ose e g) a~ld con~ol-DAH-Sepharose (lane 10)~ The balld ~n lane 11 r~p~esen~ ~e mat~al applied to the resi~s ~n ~his e~e~ent.

Pigure 5 ] nh~ on of ICA~-l ~intlin~ to Y~ontrol- a~d cont~ol-D,~H~Se~arose with ~a~lous reage~ts.

W~ole liver ICAM-l (~) in ~,he abse~ce of ~DTA w~s incubated in the prese~ce of ~e indicated reagents wi~h P~o~trol~
Sepharose, an~ ~he boulld mate~ial eluted with sr~s and analysed by immlln~blotti~g ~th the anti-EARLEC ~nl ihor1y, Shown is the amo~t of IGAM-l bound to P~ontrol-~AEI-Sepharose ~Lane 1) i~
buffer alo~le, or L~ :he presence of: S mg/ml HA (L~e 2), 5 mg/ml he~ ne 3), ~ m~/ml poly-L-glu~nic acid (la~e 4), 25 mg/ml HMBA (la~.e 53, 0~75 M a~d 1.5 M NaCl (laIIes 6 and 7, ~espe~lively).
Th~ band Lll ~e 8 rep~esents the mate~ applied t~ the resins.
Sho~n in ~ b~ is n~te~ ou~d to control-r~AH-Sepha~ose wher~
inc~ ed wi~h LEC ~ t i~ the ~bsen~e (l~e 1) or presence (lane 2~ of 5 mg~ml hep~. T~e band in L~ne 3 reyresellts ~e ~t~al applied t~ ~e resi~s.

~i~ure 6 ] ,~ n~blot~ of mater~l eluted from vqrious rP.cins w~

~x~acts of culh~red LEC in the abse~ce of EI~TA were applied to the indicated ~ esi~s in ~olum~s, a~ld the HA eluted ~ate~ analys~d with ~ullo~o~5 proh~ u~ he a~-HARLEC an~body. ~hown iS mate~ia1 el~t~l f~O~l HA-EDA-SeI h:~rOS~ (1aI1e 1)J con~ol~ A-SePharOSe ~1ane ;Z), HA-~ Sepha~Ose (1aI1e 3~ a~ld CO~1ttO1-~
Seph~rose (lane 4~. The ~d in la~le 5 rep~ese~l~s 2% of ~e applied mate~ial.

Fi~ure 7 !~l~S~PA~ autoradio~r~ph~ ~ysis of ms~t eluted fr~m var~u esing ~uth HA~

Ex~acts of 125~ surface Labelle~ LEC ir~ ~e a~se~ce (a) and presence (lb) of O.5~ TA wer~ applied to ~e inr1ic~tecl resins in colu~s, ~n~ the ~A eluted m~erial arlalysed by S~S-PAGE and autoradlo~ r~p~y. Shown is mater~al eluted f~om HA-Er~A-2 1 9394 ~

Sepharose (a l~e 1 and b lane 1)~ con~ol-EI)A-Sepha~e (a l~ne 2 and b lane ~ AH-Sepha~o~e (a l~e 3 and b lane 3) and con~ol-D~-Sepharose (a lane 4 and b laIle 4).

As many changes can be made to the embodiments without departing from the scope of the invention, it is intended that all material contained herein be interpreted as illustrative of the invention and not in a limiting sense.

Claims (24)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE AS FOLLOWS:

1. Hyaluronan bound to a compound comprising hexamethylene carrying either a terminal primary amine or a terminal acetamido group.

2. Intercellular adhesion molecule-1 (ICAM-1) bound to a compound comprising hexamethylene.

3. In a method of purifying hyaluronan binding proteins comprising combining the hyaluronan binding proteins with a resin which reacts with hyaluronan via an ethylene chain and recovering the hyaluronan binding proteins.

4. In a method of purifying hyaluronan comprising combining the hyaluronan with a compound containing a reactive ethylene chain for recovering with the hyaluronan and recovering the hyaluronan.

5. The process of claim 3 or 4 wherein the hyaluronan and hyaluronan binding proteins have molecular weights of approximately 200,000 and 400,000 daltons (protein standard).

6. Hyaluronan bound to a compound comprising 1-amino-6-acetamidohexane group.

7. The use of an effective amount of a compound having the general formula R1R2N(CH2)mNR3R4 for the inhibition of the ICAM-1, wherein R1 and R2 are selected from H and and 3 and R3 and R4 are selected from H, , . and and wherein m is an integer from 1-10.

8. The use of an effective amount of a compound having the general formula R1R2N(CH2)mNR3R4 for the inhibition of hyaluronan, wherein R1 and R2 are selected from H and and R3 and R4 are selected from H, , , and and wherein m is an integer from 1-10.

9. The use of claim 7 or 8 wherein m is an integer of 5 or greater.

10. The use of claim 7 or 8 wherein m is an integer of 6 or greater.

11. The use of claim 7, 8, 9 or 10 wherein the compound hexamethylene bis-acetamide (HMBA).

12. The use of claim 7, 8, 9, 10 or 11 wherein one of R1, R2, R3 and R4 is further selected from a drug which has been bound to the said compound.

13. A method of treatment whereby a non-toxic compound in a non-toxic effective amount of the formula or a compound containing a moiety of the formula can be administered to a mammal (e.g. human) in an effective non-toxic therapeutic amount to block the action of hyaluronan and ICAM-1 in the mammal by, for example, reacting with the compound or compounds containing moiety.

14. The method of claim 13 wherein R, R1, R2 and R3 may be hydrogen or , or while n=1, 2 or 3 or one of R, R1, R2 or R3 may be a drug combined with the moiety.

15. The method of claim 14 wherein the non-toxic compound contains at least one of R and R2 are or .

16. The method of claim 13 wherein the said non-toxic compound may have the formula R4-NH-(CH2)n-NH-R5 where 5~n~10 and R4 or R5 are each H or where R6 is CnH2n+1 where 1~n~5.

17. The method of claim 13 wherein R3 is a drug.

18. The method of claim 17 wherein the drug is selected from the following:
R= NSAIDS such as acetic acid types and propionic acid types (especially ibuprofen) anti-inflammatory gold compounds such as auranofin (Merck Index 10th Ed. no. 882) 4-aminoquinoline type drugs such as chloroquine Colchicine Vinca alkaloids such as: Vinblastine (Merck Index 10th Ed. no. 9784) Vinblastine (Merck Index 10th Ed. no. 9788) Vinblastine (Merck Index 10th Ed. no. 9789) Histamine H1-antagonist drugs of the general structure:

X=C, N
Ar=phenyl group Cyclosporin Glucocorticoid type agents Steroids Anabolic steroids such as nandrolone (Merck Index 10th Ed. no. 6211) Oestrogen type drugs such as stilboestrol Anti-androgen drugs such as cyproterone Contraceptive drugs Muscarinic antagonists such as atropine, homoatropine, cyclopentolate, tropicamide Medium duration anticholinesterase drugs such as physostigmine Histamine H2-receptor antagonists Adrenoreceptor antagonists such as propranolol, ergotamine, alprenotol, practolol, metoprolol.

19. A method of purifying hyaluronan by the use of the compound referred to in claim 13 or compound with the moiety referred to in claim 13, said method used to purify hyaluronan is accomplished by combining the compound or compound with the moiety with a solution comprising hyaluronan to permit its reaction with the hyaluronan, thereafter recovering the reaction product of the moiety and hyaluronan and thereafter releasing the compound with moiety or compound from the hyaluronan and recovering the pure hyaluronan.

20. The method of claim 19 wherein the releasing of the hyaluronan from the compound or compound containing the moiety may be accomplished by dialysis.

21. The method of claim 19 wherein the releasing of the hyaluronan is accomplished by electroelution and electrodialysis.

22. The use of claim 7 to 12 wherein a suitable effective non-toxic amount of the compound or compound containing moiety for administration to a mammal is between about 1 to 10,000µg/70kg person and preferably between about 1000 to 6000µg (1 to 6mg)/70kg person.

23. The method of claim 13 to 18 wherein a suitable effective non-toxic amount of the compound or compound containing moiety for administration to a mammal is between about 1 to 10,000µg/70kg person and preferably between about 1000 to 6000µg (1 to 6mg)/70kg person.

24. The method of claim 13 to 18 wherein the suitable compound is hexamethylene-bisacetamide (HMBA).