Inhibition of L-selectin and P-selectin mediated binding using heparin

Each of the references cited herein, including the references numbered 18, 25 and 57 to 200, is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to molecular biology and, more specifically, to methods of modulating L-selectin and P-selectin mediated binding in a subject by administering heparin to the subject in an amount that does not produce substantial anticoagulation activity or undesirable bleeding.

2. Background Information

L-selectin, E-selectin and P-selectin mediate the initial adhesive events directing the homing of lymphocytes into lymphoid organs, as well as the interactions of leukocytes and other inflammatory cells with endothelium at sites of inflammation. L-selectin is expressed on leukocytes, E-selectin is expressed on endothelium and P-selectin is expressed on platelets and endothelium. The three selectins bind to specific carbohydrate structures on opposing cells, for example, L-selectin binds to platelets and endothelium, whereas P-selectin and E-selectin bind to leukocytes.

Selectin adhesion is involved in disorders such as pathologic reperfusion injury, inflammatory disorders and autoimmune disorders. Selectin interactions also can mediate primary adhesive mechanisms involved in the metastasis of certain epithelial cancers. Thus, selectins are potential therapeutic targets for the treatment of pathologies characterized by undesirable or abnormal interactions mediated by selecting.

Much work has been directed to finding small carbohydrate molecules for use as competitive inhibitors to block selectin mediated interactions. For example, the tetrasaccharide sialyl-Lewis

x

(SLe

x

) is recognized by all three selecting, and is a component of many naturally occurring high affinity selectin ligands, for example, the myeloid cell ligand for P-selectin, called PSGL-1. However, the interaction of selectins with purified SLe

x

is weak and SLe

x

demonstrates little selectivity among the selectins. Thus, while SLe

x

and related structures may provide some therapeutic use, they are limited in being weak and nonselective inhibitors of selectin binding. Furthermore, they are very expensive to produce in the quantities required for treatment.

Heparan sulfates (HS) are naturally occurring glycosaminoglycan (GAG) chains that have diverse biological functions, generally mediated by their ability to interact with growth factors, receptors and the like. Heparin, which is a heavily modified GAG, is a heterogeneous mixture of long, unbranched carbohydrate chains consisting of repeating disaccharide units composed of uronic acids alternating with glucosamine residues which can be extensively modified.

Due to its anticoagulant activity, heparin is used clinically as an antithrombotic agent for treating human subjects having a disorder resulting from the abnormal or undesirable activation of the blood clotting cascade. Heparin's anticoagulant activity is the result of a modified pentasaccharide sequence that is present on certain heparin chains and binds to antithrombin III, a regulatory protein of the clotting cascade. Heparin chains that are longer than about 18 saccharide units and that have the modified pentasaccharide sequence can enhance the ability of antithrombin III to bind to and inhibit the function of coagulation factors, thereby inhibiting the blood clotting cascade. Pharmaceutical preparations of heparin are enriched for antithrombin III binding chains, but also contain a mixture of other components.

L-selectin and P-selectin interact with a variety of sulfated compounds including heparan sulfates porcine intestinal mucosal (PIM) heparin and its fragments. Although L-selectin and P-selectin similarly bind to HS chains and PIM-heparin, they are different from E-selectin, which fails to bind the HS chains or PIM-heparin. The observation that L-selectin binding to endothelial cell HS chains is calcium dependent indicates that this interaction is similar to selectin's interaction with natural ligands. This suggestion is supported by the ability of small heparin fragments to compete with L-selectin and P-selectin binding to SLe

x

.

Pharmaceutical preparations of heparin are similar to crude commercial PIM-heparin. Thus, it might be expected that pharmaceutical heparin would have been used for inhibiting the binding of L-selectin and P-selectin to ligands present on cells in humans. However, heparin has not been used for the purpose of inhibiting L-selectin and P-selectin binding in humans because of concerns about potential undesirable side effects associated with its anticoagulant activity.

To address the problem of undesirable anticoagulant activity, low molecular weight (LMW) oligosaccharides, which are derived from heparin, but lack anticoagulant activity, have been prepared. When injected into mice, such LMW heparins can inhibit inflammation by binding to L-selectin and P-selectin. However, the cost of preparing LMW forms of heparin combined with the cost of new product testing to obtain FDA approval for use in humans is often prohibitive. In addition, it is unclear how efficacious the LMW heparins would be in inhibiting L-selectin and P-selectin binding when administered to a human subject. Thus, a need exists for developing methods of using readily available pharmaceutical compositions to inhibit L-selectin and P-selectin mediated binding in a subject without producing undesirable side effects. The present invention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention provides a method of inhibiting L-selectin and P-selectin binding in a subject by administering to the subject an amount of heparin that does not produce substantial anticoagulant activity or undesirable bleeding. Thus, the invention provides methods of administering heparin in amounts that result in attaining a concentration less than about 0.2-0.4 units heparin per ml of plasma in the subject, such levels which inhibit L-selectin or P-selectin mediated binding in the subject. The invention further provides methods of treating a subject having an L-selectin or P-selectin related pathology by administering heparin in an amount that does not produce substantial anticoagulant activity or undesirable bleeding, for example, an amount that results in a concentration less than about 0.2-0.4 units heparin per ml of plasma in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A

shows that heparin sulfate obtained from endothelial cells binds to L-selectin.

FIG. 1B

shows that heparin sulfate obtained from endothelial cells does not bind to E-selectin.

FIG. 1C

shows that heparin sulfate obtained from endothelial cells binds to P-selectin.

FIG. 2A

shows that the binding of heparin sulfate glycosaminoglycan chains (HS-GAG) by L-selectin is calcium-dependent.

FIG. 2B

shows that binding of heparin sulfate glycosaminoglycan chains (HS-GAG) by P-selectin is not calcium-dependent.

FIG. 3

shows that the binding of the HS-GAG chains to P-selectin does not require exogenously added cations. HS-GAG in buffer lacking exogenously added cations was applied to a P-selectin column, washed with the same buffer and eluted with 20 mM EDTA buffer. Arrows as in FIG.

1

.

FIG. 4A

shows that fractions of porcine intestinal mucosal (PIM) heparin do not bind to E-selectin.

FIG. 4B

shows that fractions of porcine intestinal mucosal (PIM) heparin can bind to L-selectin.

FIG. 4C

shows that fractions of porcine intestinal mucosal (PIM) heparin can bind to P-selectin.

FIG. 5A

shows the results of experiments in which the binding of PIM-heparin to L-selectin was shown to be calcium dependent.

FIG. 5B

shows the results for PIM-heparin applied to a P-selectin affinity column. In both of these experiments, the PIM-heparin was applied in a magnesium/EGTA buffer lacking exogenously added calcium.

FIGS. 6A

to

6

D show the size dependence of porcine mucosal heparin fragments in binding to an L-selectin affinity column.

FIG. 6A

shows the results for octasaccharides,

FIG. 6B

shows the results for decasaccharides,

FIG. 6C

shows the results for dodecasaccharides, and

FIG. 6D

shows the results for tetrasaccharides. In these experiments, the columns were eluted with 2 mM EDTA (“2”), followed by 20 mM EDTA (“20”) buffer.

FIGS. 7A

to

7

D show the results of rechromatography of PIM-heparin tetradecasaccharides on an L-selectin affinity column.

FIG. 7A

shows the results for tetradecasaccharides that originally were unbound (Pool A),

FIG. 7B

shows the results for tetradecasaccharides that were slightly retarded,

FIG. 7C

shows the results for tetradecasaccharides that were retarded, and

FIG. 7D

shows the results for tetradecasaccharides that were eluted with EDTA.

FIG. 8A

shows the inhibitory properties of a pharmaceutical heparin preparation (unfractionated; closed triangles) or LMW heparins (LOVENOX, open squares; FRAGMIN, closed circles) on the binding of L-selectin to immobilized SLe* using ELISA inhibition experiments.

FIG. 8B

shows the inhibitory properties of a pharmaceutical heparin preparation (unfractionated; closed triangles) or LMW heparins (LOVENOX, open squares; FRAGMIN, closed circles) on the binding of P-selectin to immobilized SLe* using ELISA inhibition experiments.

FIG. 9

shows the inhibition of HL-60 cell attachment to immobilized L-selectin (open squares) or P-selectin (closed circles) by a pharmaceutical heparin preparation (unfractionated). The horizontal bar indicates the recommended plasma concentration range for therapeutic use of heparin as an anticoagulant in a human subject.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of inhibiting L-selectin or P-selectin mediated binding in a subject by administering to the subject an amount of heparin that does not produce substantial anticoagulant activity or undesirable bleeding in the subject. Heparin is used clinically as an anticoagulant to reduce blood clotting in an individual. Heparin has a narrow therapeutic window and is administered to individuals for anticoagulant therapy in an amount sufficient to achieve levels of 0.2-0.4 units per ml of plasma (Ginsberg,

New Engl. J. Med.

335:1816-1828 (1996)). Individuals administered heparin at concentrations less than 0.2 units per ml of plasma are significantly more likely to exhibit a recurrence of thrombosis, indicating that administration of an amount of heparin that results in a concentration of less than about 0.2 units per ml of plasma generally is not optimal for anticoagulant therapy of an active thrombus.

As disclosed herein, heparin, as formulated for clinical use, can inhibit the binding of P-selectin and L-selectin to their ligands. Significantly, approximately one-fifth to one-tenth of the amount of pharmaceutical heparin that is needed for anticoagulant therapy in humans effectively inhibits L-selectin and P-selectin mediated binding. Thus, the invention provides a means to inhibit L-selectin and P-selectin mediated binding in a subject by administering heparin in an amount that does not produce substantial anticoagulant activity or undesirable bleeding in the subject.

The amount of heparin administered to a subject to inhibit L-selectin or P-selectin binding is characterized in that it does not produce undesirable bleeding as a side effect, although it can produce mild anticoagulant activity. As a result, side effects such as bleeding complications that are associated with using heparin for anticoagulant therapy are not a concern. The amount of heparin administered in a method of the invention results in a concentration of less than about 0.2 to 0.4 units heparin/ml plasma, generally about 0.1 to 0.2 units heparin/ml plasma, or about 0.05 to 0.1 units heparin/ml plasma, and can be about 0.02 units heparin/ml plasma or less.

The invention additionally provides a method of inhibiting P-selectin mediated binding, but not L-selectin mediated binding, in a subject by administering heparin in an amount that results in a concentration of about 0.02 to 0.05 units heparin/ml plasma, preferably about 0.005 to 0.02 units heparin/ml plasma to the subject. For example, an amount of heparin that results in about 0.002 units heparin/ml plasma can be administered to a subject to inhibit P-selectin mediated binding, whereas such a concentration of heparin does not substantially inhibit L-selectin mediated binding.

Although the ability of heparin to inhibit L-selectin and P-selectin binding has been established, its anticoagulant activity has prevented clinicians from using heparin to inhibit L-selectin and P-selectin binding-in a subject (Yednock et al.,

J. Cell Biol.

104:713-723 (1987); Skinner et al.,

Biochem. Biophys. Res. Commun.

164:1373-1379 (1989); Aruffo et al.,

Cell

67:35-44 (1991); Handa et al.,

Biochem. Biophys. Res. Commun.

181:1223-1230 (1991); Skinner et al.,

J. Biol. Chem.

266:5371-5374 (1991); Needham and Schnaar,

Proc. Natl. Acad. Sci. USA

90:1359-1363 (1993); Nelson et al.,

Blood

82:3253-3258 (1993); Yuen, et al.,

J. Biol. Chem.

269:1595-1598 (1994); Mitsuoka et al.,

Biochem. Biophys. Res. Commun.

230:546-551 (1997); Yoshino et al.,

J. Med. Chem.

40:455-462 (1997); Norgard-Sumnicht et al.,

Science

261:480-483 (1993)). Thus, molecules that can inhibit L-selectin and P-selectin binding without exhibiting anticoagulant activity have been identified. For example, Bevilacqua et al. demonstrated that particular low molecular weight fractions of heparin which lack anticoagulant activity modulate selectin binding (U.S. Pat. No. 5,527,785, issued Jun. 18, 1996, which is incorporated herein by reference). Bevilacqua et al. also used crude nonpharmaceutical grade heparin to inhibit selectin binding in vitro. However, Bevilacqua et al. stated that heparin compositions that contain the pentasaccharide antithrombin III binding sequence, which is present in pharmaceutical heparin preparations, should not be used for inhibiting selectin mediated binding in a subject due to its anticoagulant activity.

As disclosed herein, pharmaceutical preparations of heparin inhibit L-selectin and P-selectin mediated binding at concentrations less than about 0.2-0.4 units heparin per ml of plasma. This amount of heparin can produce mild anticoagulant activity in humans, but usually does not produce undesirable bleeding. For example, two separate lots of unfractionated pharmaceutical heparin significantly inhibited L-selectin and P-selectin binding at concentrations less than the recommended therapeutic levels targeted for anticoagulation therapy in a subject. In particular, the concentration of pharmaceutical heparin needed to inhibit 50% of the binding (IC

50

) of SLe

x

to L-selectin and P-selectin was 0.07-0.08 units/ml and 0.01-0.02 units/ml, respectively (see Table 5, below). In addition, IC

50

values for pharmaceutical heparin to inhibit attachment of HL-60 cells, which express the natural selectin ligand PSGL-1, to immobilized selectins were 0.02-0.03 units/ml for L-selectin and 0.003-0.01 units/ml for P-selectin (Table 5). Thus, clinically approved heparin compositions can inhibit L-selectin and P-selectin mediated binding in an amount that does not produce substantial anticoagulant activity or undesirable bleeding in a subject.

The methods of the invention are useful in treating a subject having an L-selectin or P-selectin related pathology. Many pathological processes involve L-selectin and P-selectin (Table 1) and, as disclosed herein, can be treated by administering heparin in an amount that results in a concentration of less than about 0.2-0.4 units/ml of plasma in a subject. Ischemia and reperfusion, for example, which cause significant tissue injury in clinical disorders such as stroke, myocardial infarction, organ transplantation and organ hypoperfusion, involve L-selectin and P-selectin related processes (Seekamp et al.,

Am. J. Pathol.

144:592-598 (1994); Garcia-Criado et al.,

J. Am. Coll. Surgeons

181:327-334 (1995); Moore et al.,

J. Appl. Physiol.

78:2245-2252 (1995); Mihelcic et al.,

Blood

84:2322-2328 (1994); Han et al.,

J. Immunol.

155:4011-4015; Rabb et al.,

Am. J. Physiol. Renal Fluid Electrolyte Physiol.

271:F408-F413 (1996); Buerke et al.,

J. Clin. Invest.

93:1140-1148 (1994); Miura et al.,

Ann. Thorac. Surg.

62:1295-1300 (1996); Flynn et al.,

Am. J. Physiol. Heart Circ. Physiol.

271:H2086-H2096 (1996); Ma et al.,

Circulation

88:649-658 (1993); Buerke et al.,

J. Pharmacol. Exp. Ther.

271:134-142 (1994); Lefer,

Ann. Thorac. Surg.

60:773-777 (1995); Haught et al.,

Am. Heart J.

132:1-8 (1996); Turunen et al.,

J. Exp. Med.

182:1133-1141 (1995); Morikawa et al.,

Stroke

27:951-955 (1996); Weiser et al.,

Shock

5:402-407 (1996); Yamadia et al.,

Blood

86:3487-3492 (1995); Kubes et al.,

Am. J. Physiol. Heart Circ. Physiol.

267:H931-H937 (1994); Gibbs et al.,

Surgery

119:652-656 (1996); Takada et al.,

J. Clin. Invest.

99:2682-2690 (1997); Zizzi et al.,

TABLE 1

TISSUE

PATHOLOGY

REFERENCE

Pathological processes involving L-selectin

Lung

Homing of eosiniphils to asthmatic lungs

(58, 59)

Acute lung injury

(60, 61, 61-64)

Inflammation

(65, 66)

Ischemia-reperfusion injury

(67, 68)

Sepsis-induced lung injury

(69)

Adult respiratory distress syndrome

(57)

(ARDS)

Multi-

Inflammatory injury

(70, 70-72)

system

Ischemia-reperfusion injury

(67)

Immune

HIV infection

(73)

Impaired primary T-cell response

(74)

Common variable immunodefiency

(75)

(CVID)

Chronic myelocytic leukemia

(76)

Primary Sjogren's syndrome

(77)

Inflamed extralymphoid tissues

(78)

Humoral immune response (in spleen)

(79)

Meningeal leukemia

(80)

Pancreas

Nonobese diabetes

(81-83)

Skeletal;

Rheumatoid arthritis

(84)

Connective

Anklosing spondylitis

(85)

tissue

System schlerosis

(85)

Vasculitis

(85)

Muscular

Ischemia-reperfusion injury

(67)

Liver

Primary infection with

Listeria

(86)

monocytogenes

Liver inflammation

(87)

Skin

Cutaneous inflammation

(88)

Ischemia-reperfusion injury

(89, 90)

Systemic inflammatory response syndrome

(91, 92)

(SIRS)

Gut

Inflammatory injury

(93)

Inflamed peritoneum

(94)

Colon carcinoma

(95)

Kidney

Allograph rejection

(25)

Impaired granulocyte function in patients

(96)

with chronic hemodialysis

Renal ischemia-reperfusion injury

(97)

Circulatory

Inflames/injured venular endothelium

(98, 99)

Ischemia-reperfusion injury

(100-106)

PMN leukocyte-induced vasocontraction

(107)

and endothelial dysfuntions

Hemorrhagic shock injury

(108)

Peripheral arterial disease

(109)

Cardiac transplant rejection

(110)

Activated endothelium

(111)

CNS

Pleocytosis in bacterial meningitis

(112)

Allergic encephalomyelitis

(113)

Meningoencephalitis

(114)

Ischemia-reperfusion injury

(115)

Multiple sclerosis (MS)

(116)

Misc.

Failure of tumor immunity

(117)

Plasmodium falciparium malaria

(118)

Increased soluble L-selectin in premature

(119, 120)

infants

Burn injury

(57)

Hyperthyrodism

(121)

Pathological processes involving P-selectin

Lung

Acute lung injury

(60, 63, 64,

122-127)

Reduction of allergic airway hyper-

(128)

responsiveness

Ischemia-reperfusion injury

(67)

Inflammation induced by Streptococcus

(66)

pneumonia

Hyperoxic lung injury

(129)

Small cell lung carcinoma

(130)

Adult respitory distress syndrome (ARDS)

(57)

Asthma

(131)

Pleurisy

(132)

Multi-

Inflammatory injury

(18, 72, 133)

system

Sepsis and organ failure due to severe

(134)

trauma

Burn injury

(57, 135)

Ischemia-reperfusion injury

(67)

Immune

Leukocyte adhesion syndrome, type II

(136)

(LAD II)

Infection susceptibility

(137)

Primary Sjogren's Syndrome

(77)

Arthus reaction

(138)

Lack of recruitment of Helper T-1 cells to

(139, 140)

inflammatory sites

Peripheral neutrophilia

(141)

Altered serum selectin levels in leukemia

(142)

patients

Altered hematoiesis and infection

(137, 143)

susceptibility

Severe trauma, sepsis, organ failure

(134)

Pancreas

Nonobese diabetes

(144)

Skeletal;

Rheumatoid arthritis

(84, 145, 146)

Connective

tissue

Muscular

Skeletal muscle ischemia-reperfusion

(147)

injury

Injury and inflammation to striated muscle

(148)

Liver

Ischemia-reperfusion injury

(149)

Improved liver function after hemorrhagic

(150)

shock

Skin

Cutaneous inflammation

(88, 148, 151)

Ear reperfusion injury

(90, 152)

Ischemia-reperfusion injury in dorsal skin

(153)

Delayed-type contact hypersensitivity

(92, 154)

(DTH)

Wound Repair

(155)

Gut

Intestinal mucosal injury

(156, 157)

Inflammatory injury

(158)

Early phase histamine-induced

(159)

inflammation

Ischemia-reperfusion injury

(160)

Inflamed peritoneum

(94)

Intestinal ischemia-reperfusion injury

(161)

Inflammatory bowel disease

(162)

Acute injury to peritoneum

(163)

Abdominal inflammation

(164)

Colon carcinoma

(95, 165)

Intestinal inflammation

(166)

Kidney

Neutrophil-dependent glomerular injury

(167)

Acute passive antiglomerular basement

(114)

membrane nephritis

Glomerulonephritis

(168)

Ischemia-reperfusion injury

(169, 170)

Circulatory

Inflamed/injured venular endothelium

(171)

Splanchnic ischemia-reperfusion injury

(172, 173)

Myocardial ischemia-reperfusion injury

(100-102, 105,

174, 174-178,

178, 179)

PMN leukocyte-induced vasocontraction

(107)

and endothelial dysfunctions

Severe vascular trauma

(180)

Atherogenesis brought on by oxidized

(181)

LDL

Inflammation and thrombosis

(182)

Hemorrhagic shock injury

(183)

Reduced thrombis formation

(184, 185)

Atherosclerotic vascular disease

(186)

Disrupted blood hemostasis

(187)

Increased infection susceptibility

(143)

Altered hematopoiesis

(143)

Grey platelet syndrome

(188)

Peripherial arterial disease

(109)

Endothelial cell hypoxia and hypoxia

(189, 190)

regeneration

Enhanced heart allograft afteriosclerosis

(191)

Hypercholesterolemia

(192)

Angina pectoris/Unstable angina

(193, 194)

Coronary spasm

(195)

Vasocontraction and endothelial cell

(107)

dysfunction

Atherogenesis and hypertension

(186)

CNS

Ischemia-reperfusion injury

(115)

Brain meningitis

(196)

Eye

Intraocular inflammation

(197)

Misc.

Hypertension of the adrenal glands

(198)

Increased expression of P-selectin in

(199)

breast cancer tissue

Hyperthyroidism

(121)

Tumor angiogenesis

(200)

J. Pediatr. Surg.

32:1010-1013 (1997); Davenpeck et al.,

Am. J. Physiol. Heart Circ. Physiol.

267:H622-H630 (1994); Gauthier et al.,

Am. J. Physiol. Gastrointest. Liver Physiol.

267:G562-G568 (1994); Weyrich et al.,

J. Clin. Invest.

91:2620-2629 (1993); Tojo et al.,

Glycobiology

6:463-469 (1996); Lefer et al.,

Am. J. Physiol. Heart Circ. Physiol.

271:H2421-H2429 (1996)). Thus, such disorders can be treated using a method of the invention such that the severity of the pathology is reduced. Similarly, acute and chronic inflammatory disorders can be treated using a method of the invention.

For the methods of the invention, an amount of heparin that does not produce substantial anticoagulant activity or undesirable bleeding is administered to the subject. As used herein, reference to “an amount of heparin that does not produce substantial anticoagulant activity” means an amount of heparin that does not cause bleeding complications, although a mild anticoagulant effect can occur. Thus, the amount of heparin administered generally will result in a plasma heparin level of less than about 0.2-0.4 units/ml plasma in a subject.

Clinical signs and symptoms of undesirable bleeding includes blood in the urine, or stool, heavier than normal menses, nose bleeds or excessive bleeding from minor wounds or surgical sites. Easy bruising can precede such clinical manifestations. Where undesirable bleeding occurs, heparin activity can be neutralized by administration of protamine sulfate.

Although an amount of heparin administered to inhibit L-selectin and P-selectin mediated binding in a subject will depend, in part, on the individual, normal adult subjects administered heparin in amounts that result in less than 0.2 units heparin/ml of plasma generally do not exhibit undesirable bleeding. A subject treated with heparin can be monitored for undesirable bleeding using various assays well known in the art. For example, blood clotting time, active partial thromboplastin time (APTT), or anti-Xa activity can be used to determine if coagulation status is undesirably increased in a subject administered heparin. Where undesirable bleeding occurs, heparin administration is discontinued.

The amount of heparin administered depends, in part, on whether L-selectin or P-selectin mediates binding and, therefore, whether only P-selectin, or both L-selectin and P-selectin, are to be inhibited. For example, an amount of heparin less than that used for anticoagulant therapy can be administered to a subject for the purpose of substantially inhibiting P-selectin as compared to L-selectin (see Table 5 and FIGS.

8

and

9

). The amount of heparin administered to a subject also depends on the magnitude of the therapeutic effect desired.

In addition, the amount of heparin administered will depend on the individual subject because the bioavailability of heparin within subjects is known to vary. For example, heparin dosages are sometimes administered in units heparin/kg body weight. However, the dosages of heparin needed (e.g. units heparin/kg body weight) to attain specific levels of heparin in the plasma of a subject can vary among individuals because of differences in heparin bioavailability. Thus, the heparin concentration in the blood of a subject in units/ml plasma is the more reliable measure of heparin concentration. The amount of plasma heparin in a subject can be determined using titration and neutralization assays with protamine sulfate.

Heparan sulfate (HS) chains released from endothelial cell heparan sulfate proteoglycans (HSPGs) bind to L-selectin (Norgard-Sumnicht and Varki,

J. Biol. Chem.

270:12012-12024 (1995)). Selectin-Receptor globulin (Rg) chimeric proteins were used to make affinity columns to determine if these HS ligands also bound to other selectins (P and E). Metabolically labeled HS chains obtained from two endothelial cell lines were applied to each selectin affinity column (see Example I). HS ligand bound primarily to the L-selectin and P-selectin column, but not to the E-selectin column (see FIG.

1

). The HS ligand elution profiles using 5 mM EDTA buffer were different; all ligand was released from L-selectin, whereas there was poor release of ligand from P-selectin (see

FIG. 1C

, inset). Thus, a P-selectin affinity column completely bound the L-selectin HS ligands from both endothelial cell types, but showed a comparatively slow and partial elution with 5 mM EDTA. Addition of 20 mM EDTA resulted in recovery of all of the labeled ligand from both cell sources. This difference in HS elution between the L-selectin and P-selectin columns indicates that the eluting effect of EDTA with P-selectin might not be strictly based upon its calcium chelating properties (see below).

High affinity interactions of selectin generally are dependent on calcium. In addition to calcium, magnesium can potentiate the effects of calcium in P-selectin binding (Geng et al.,

Nature

343:757-760 (1990)). However, the HS ligands studied here are structurally different from the previously described sialylated ligands (Varki,

Proc. Natl. Acad. Sci. USA

91:7390-7397 (1994); Furie and Furie,

Thromb. Haemost.

74:224-227 (1995); Vestweber,

J. Clin. Invest.

98:1699-1702 (1996); McEver et al.,

J. Biol. Chem.

270:11025-11028 (1995); Nelson et al,

Annu. Rev. Cell Biol.

11:601-631 (1995); Springer,

Annu. Rev. Physiol.

57:827-872 (1995); Crocker and Feizi,

Curr. Opin. Struct. Biol.

6:679-691 (1996); Kansas,

Blood

88:3259-3287 (1996); Rosen and Bertozzi,

Curr. Biol.

6:261-264 (1996); Vestweber,

J. Cell. Biochem.

61:585-591 (1996); Varki,

J. Clin. Invest.

99:158-162 (1997); Phillips et al.,

Science

250:1130-1132 (1990); Walz et al.,

Science

250:1132-1135 (1990); Polley et al.,

Proc. Natl. Acad. Sci. USA

88:6224-6228 (1991); Berg et al.,

J. Biol. Chem.

266:14869-14872 (1991); Tyrell et al.,

Proc. Natl. Acad. Sci. USA

88:10372-10376 (1991); Berg et al.,

Biochem. Biophys. Res. Commun.

184:1048-1055 (1992); Foxall et al.,

J. Cell Biol.

117:895-902 (1992); Larkin et al.,

J. Biol. Chem.

267:13661-13668 (1992); Rosen and Bertozzi,

Curr. Opin. Cell Biol.

6:663-673 (1994)) and, therefore, an analysis of the calcium dependence of heparan sulfate chain binding to L-selectin and P-selectin was performed.

The calcium dependence of L-selectin binding was determined using binding buffer without added calcium. The binding was not changed, although trace amounts of calcium (approximately 50 μM) present in a magnesium-containing buffer may support L-selectin binding. Indeed, binding of HS to L-selectin was abolished in magnesium/EGTA buffers, in which calcium was lowered to negligible amounts (FIG.

2

A). In contrast, binding of the HS chains to P-selectin occurred in the magnesium/EGTA buffer (FIG.

2

B), indicating that HS binds to P-selectin in the absence of exogenously added calcium.

FIG. 3

further demonstrates that binding by P-selectin in the absence of exogenously added cations still requires 20 mM EDTA buffer for elution of the HS chains. These results show that although the binding specificities of L-selectin and P-selectin overlap, there are differences, including the different requirement for calcium. However, the presence of calcium in vivo indicates that heparin will inhibit the binding of L-selectin in a subject.

The need for 20 mM EDTA to elute HS chains from the P-selectin column in the absence of divalent cations indicates that either trace amounts of calcium remain tightly bound to the selectin column from the prior experiments or that EDTA is eluting the ligand from P-selectin by a mechanism unrelated to its divalent cation chelating properties. However, it is unlikely that trace amounts of calcium remain tightly bound to the selectin column because crystal structures of E-selectin and the related C-type lectin mannose binding protein indicate that the calcium ion is exposed to the solvent and does not lie deeply buried in the binding pocket (Graves et al.,

Nature

367:532-538 (1994); Weis et al.,

Nature

360:127-134 (1992)). More likely, EDTA is eluting the ligand from P-selectin by a mechanism unrelated to its divalent cation chelating properties because an equivalent (80 mM) increase in chloride concentration did not cause elution. Thus, EDTA can elute the HS ligands due to its inherent polycarboxylate structure, for example, by mimicking the high charge density of HS chains.

HS chains are available in limited quantities so the ability of commercially available mast cell derived PIM-heparin to bind to the selecting was examined (Example II). In addition, PIM-heparin is used as an anticoagulant in humans and its potential use as a selectin therapeutic was of interest. Labeled PIM-heparin in a column buffer adjusted to more closely approximate physiological conditions (Koenig et al.,

Glycobiology

7:79-93 (1997)) was applied to each of the three selectin affinity columns and the bound material was eluted (FIG.

4

).

Similar to the results obtained for HS chains, none of the PIM-heparin molecules bound to E-selectin (FIG.

4

A). 90% of the total PIM-heparin loaded bound to L-selectin; 58% was eluted with 2 mM EDTA and 32% was eluted with 20 mM EDTA (FIG.

4

B). In contrast, 79% of the PIM-heparin bound to P-selectin, very little was eluted with 2 mM EDTA but 20 mM EDTA eluted all of the bound heparins (FIG.

4

C). These results demonstrate that the binding of PIM-heparin to the selecting was similar to the HS chains obtained from endothelial cells, except that a portion of PIM-heparin remained bound to L-selectin after adding 2 mM EDTA. Thus, heparin compositions commonly formulated for use as anticoagulants have the ability to bind to L-selectin and P-selectin.

The calcium dependence of PIM-heparin binding to L-selectin and P-selectin was similar to the binding of the endothelial HS chains to the selectins (Example II and FIG.

5

). Binding to L-selectin was abolished in magnesium/EGTA buffers (FIG.

5

A), while a significant fraction continued to bind to P-selectin under these conditions (FIG.

5

B). However, the fraction that bound to P-selectin was lower than that-seen under calcium-replete conditions (45% vs. 79%), possibly indicating a partial calcium dependency of the initial binding event. Of the fraction that bound in the absence of calcium, 20 mM EDTA was required for elution (FIG.

5

B). These results indicate that calcium occupancy in the lectin site may assist the initial binding of some heparin fragments to P-selectin. However, once bound, calcium does not appear to be required to maintain the interaction between the heparin chains and P-selectin.

PIM-heparin chains have a large molecular weight (average 20 kDa; approximately 38-40 disaccharide units) and are polydisperse in structure (Pervin et al.,

Glycobioloay

5:83-95 (1995)). In order to determine the specificity of interaction between L-selectin and heparin, the binding of size-fractionated heparin fragments to an t-selectin column was examined (Example III). The binding of PIM-heparin fragments to L-selectin and P-selectin was size dependent; substantial binding occurred with tetradecasaccharides (see FIG.

6

D and Table 2). These results indicate that the fraction of PIM-heparin that binds to L-selectin and P-selectin predominantly comprises tetradecasaccharides or larger fragments.

Heparin tetrasaccharides interact with L-selectin in blocking binding to immobilized sialyl-Lewis

x

(SLe

x

; Siao2-3 Galβ1-4(Fuca1-3)GlcNAc), a well known component of natural selectin ligands (Nelson et al., supra, 1993). Studies of other oligosaccharide protein interactions indicate that binding constants in the low μM range would be needed for the detection of selectin binding (Varki and Kornfeld,

J. Biol. Chem.

TABLE 2

Unfractionated and LMW Heparins as Inhibitors of L- and P-Selectin binding to Immobilized SLc

x

and to

[

3

H]HL-60 cells.

Therapeutic

IC

50

Units/ml*

range

Against immobilized SLc

x

Against HL-60 cells

Clinical heparin

(Units/ml)*

E-Selectin

L-Selectin

P-Selectin

L-Selectin

P-Selectin

Unfractionated Heparin

0.2-0.4

>50

0.07-0.08

0.01-0.02

0.02-0.03

0.003-0.01

Levonox (LMW Heparin, Enoxaparin)

0.6-1.0

>50

0.8-1.5

0.8-1.0

1.5-3.0

0.7

Fragmin (LMW Heparin, Deltaparin)

0.6-1.0

>50

0.7-2.0

1.5-2.0

4.0-7.0

1.0-4.0

TABLE 3

Heparin Fragment Mixtures as Inhibitors of L- and P-Selectin binding

to Immobilized SLc

x

.

IC

50

value

L-Selectin

P-Selectin

Tetradecasaccharides

54

uM

82

uM

Dodecasaccharides

159

uM

159

uM

Decasaacharides

>1900

uM

>1900

uM

Octasaccharides

>2400

uM

>2400

uM

Sialyl Lewis

x

600

uM

520

uM

Size-fractionated PIM-heparin mixtures were tested for their ability to inhibit selectin binding to immobilized Sle

x

in ELISA inhibition experiments, as described under “Materials and Methods.” There was no inhibition of E-selection, even with the tetradecasaccharides, at concentration as high as 5 mg/ml (>1000 μM). IC

50

values for L- and P-selectin inhibition were determined by the equation: {[(average of duplicates)−(average of negative controls)]/[(average of positive controls)−(average of negative controls)]}*100, where the positive controls were without inhibitors, and the negative controls contained 5 mM EDTA. Experiments were performed 2-3 times, and the averaged IC

50

values are presented. For calculation of concentration, the following average molecular weights were assumed: tetradecasaccharide, 3675 Da; dodecasaccharide, 3150 Da; decasaccharide, 2625 Da; and octasaccharides, 2100 Da. The inhibitory potency of the tetrasaccharide Sialyl Lewis

x

under the same conditions is shown for comparison.

With the availability of a size defined fraction, the binding of some molecules and the lack of binding of others indicates a degree of specificity. Such specificity was confirmed by performing rebinding experiments with the tetradecasaccharide pooled fractions were performed (Example III). For both L-selectin and P-selectin, the binding of the fractions was reproducible (see

FIG. 7

, for L-selectin, and Table 4). The divalent cation dependence of this interaction also was similar to that of intact heparin and the endothelial HS chains. In addition, the interactions with L-selectin were disrupted by 2 mM EDTA (FIG.

7

), whereas those with P-selectin required 20 mM EDTA for disruption (Table 4) and studies with magnesium/EGTA buffers confirmed the calcium dependency of L-selectin binding. These results demonstrate that various ligand populations differ over a wide range in their ability to interact with L-selectin. Thus, individual heparin chains or pools of related chains are likely to exhibit differential affinity for selectins. Such ligands that selectively bind are useful for the selective targeting of selectins in vivo.

In an effort to determine if there are differences between the types of tetradecasaccharides that are recognized by L-selectin and P-selectin, cross-binding studies were performed (Example III). Of the tetradecasaccharides that bound to L-selectin, 100% bound to P-selectin and were eluted with 20 mM EDTA (see Table 4). In contrast, not all of the tetradecasaccharides that bound to P-selectin also bound to L-selectin. Of the tetradecasaccharides from the P-selectin column that bound to L-selectin, 22% were from the 2 mM EDTA eluted fraction and 51% were from the 20 mM EDTA eluted fraction. Of the fraction that did not bind

TABLE 4

Rebinding and cross-binding of Tetradecasaccharides to

L- and P-selectin

Original Binding of sample

L-selectin

P-selectin

P-selectin

2 mM

2 mM

20 mM

Secondary

L-selectin

EDTA

P-selectin

EDTA

EDTA

Binding

unbound

eluted

unbound

eluted

eluted

L-selectin

Unbound

98%

0%

100%

78%

49%

2 mM EDTA

2%

100%

0%

22%

51%

eluted

P-selectin

Unbound

88%

0%

88%

0%

0%

2 mM EDTA

0%

0%

5%

100%

0%

eluted

20 mM

12%

100%

7%

0%

100%

EDTA

Eluted

[

3

H]labeled tetradeccasaccharides were fractionationed by P- and L-selected affinity chromatography as described under “Materials and Methods”). After desalting, aliquots of each friction were reapplied to the same column, or to the other selectin column (see

FIG. 7

for an example of the results). to L-selectin, 12% re-bound to P-selectin. These data indicate that, although both L-selectin and P-selectin exhibit some degree of selectivity, L-selectin has a greater degree of selectivity than P-selectin in binding heparin tetradecasaccharides. Thus, in view of this greater selectivity, L-selectin can be targeted over P-selectin using particular tetradecasaccharide fractions.

Since the size of the recognized tetradecasaccharides is not a variable, the differences in affinity are a result of structural differences between them. The structures of the tetradecasaccharides were examined using enzymes or chemical modification they cleave the chains at particular sites (Lohse and Linhardt,

J. Biol. Chem.

267:24347-24355 (1992); Conrad,

In Current Protocols in Molecular Biology

17-22A (1996); Desai et al.,

Biochemistry

32:8140-8145 (1993); Jandik et al.,

Glycobiology

4:289-296 (1994); Linhardt,

In Current Protocols in Molecular Biology

17-18B (1996)). Bound and flow-through material from the P-selectin and L-selectin columns were cleaved, and the products analyzed by FPLC (Example III). L-selectin binding fragments were more heavily sulfated and epimerized and, similar to the endothelial HS chains, they were enriched in free amino groups. The P-selectin binding component included this fraction as well as some less highly modified regions; they were sensitive to Heparin Lyase III. These results indicate that, while distinct structural features selectively enhance interactions of P-selectin and L-selectin with heparin tetradecasaccharides, the binding represents a continuum of affinities with no apparent structural motif being markedly superior in its ability to be recognized.

The clinical relevance of the observations made using commercially available PIM-heparin, which is very similar to the heparin used as an anticoagulant (Ginsberg,

N. Engl. J. Med.

335:1816-1828 (1996); Hirsh,

Semin. Thromb. Hemost.

22 Suppl: 7-12 (1996); Pineo and Hull,

Annu. Rev. Med.

48:79-91 (1997)) were analyzed further. ELISA inhibition studies using immobilized SLe

x

indicated that crude commercial PIM-heparin had an IC

50

of 18 μg/ml and 2 μg/ml for SLe

x

binding to L-selectin and P-selectin, respectively. Although crude heparin preparations vary between lots, the IC

50

values for crude PIM-heparin roughly corresponds to pharmaceutical heparin preparations of approximately 0.18-0.09 units/ml (L-selectin) and 0.02-0.01 units/ml (P-selectin). These results indicate that the inhibition of binding can occur at therapeutically relevant concentrations.

In order to understand the relevance of these studies to clinical practice, the effect of pharmaceutical heparin preparations on selectin binding was determined (Example IV). ELISA inhibition assays (see FIG.

8

and Table 5) demonstrated that two separate lots of pharmaceutical unfractionated heparin significantly inhibited L-selectin and P-selectin, but not E-selectin, binding at concentrations less than that recommended for anticoagulation therapy (Table 5). The calculated IC

50

values towards SLe

x

binding were 0.07-0.08 units/ml for L-selectin and 0.01-0.02 units/ml for P-selectin (Table 5).

Similar findings were obtained in an HL-60 cell attachment assay, which measured the interaction of L-selectin and P-selectin with the natural selectin ligand PSGL-1 expressed by the HL-60 cells (Example IV). PSGL-1 is a primary selectin ligand with significant

TABLE 5

Unfractionated and LMW Heparins as Inhibitors of L- and P-Selectin binding to Immobilized SLc

x

and to

[

3

H]HL-60 cells.

Therapeutic

IC

50

Units/ml*

range

Against immobilized SLc

x

Against HL-60 cells

Clinical heparin

(Units/ml)*

E-Selectin

L-Selectin

P-Selectin

L-Selectin

P-Selectin

Unfractionated Heparin

0.2-0.4

>50

0.07-0.08

0.01-0.02

0.02-0.03

0.003-0.01

Levonox (LMW Heparin, Enoxaparin)

0.6-1.0

>50

0.8-1.5

0.8-1.0

1.5-3.0

0.7

Fragmin (LMW Heparin, Deltaparin)

0.6-1.0

>50

0.7-2.0

1.5-2.0

4.0-7.0

1.0-4.0

Several clinical lots of Unfractionated and LMW Heparins were tested for their ability to inhibit selectin binding to immobilized Sle

x

in ELISA inhibition experiments or HL-60 cells binding to immobilized selectins, as described under “Material and Methods.” IC

50

values were determined by the equation: {[(average of duplicates)−(average of negative controls)]/[(average of positive controls)−(average of negative controls)]}×100, where the positive controls were without inhibitors, and the negative controls contained 5 mM EDTA in the ELISA inhibition experiments, and 20 mM EDTA in the HL-60 cell inhibition experiments. Experiments were performed 2-3 times, and the range of measured IC

50

values are presented. Examples of the results can be seen in

FIGS. 8 and 9

. Unfractionated heparin concentrations are reported in Protamine neutralization Units while LMW heparins are reported as anti-Xa Units (see text for discussion). biological relevance in vivo (Kansas, supra, 1996; Rosen and Bertozzi, supra, 1996; Vestweber, supra, 1996; Lowe and Ward,

J. Clin. Invest.

99:822-826 (1997); Varki, supra, 1997; McEver and Cummings,

J. Clin. Invest.

100:485-492 (1997)). The binding of HL-60 cells to L-selectin and to P-selectin was inhibited by unfractionated pharmaceutical heparin (FIG.

9

). The IC

50

values calculated for HL-60 binding to L-selectin and P-selectin were approximately 12-fold and 50-fold lower, respectively, than the recommended range for anticoagulant therapy (Table 5).

Pharmaceutical preparations of low molecular weight (LMW) heparins different from those described by Bevilacqua et al. have been used for anticoagulant therapy in place of unfractionated heparin due to enhanced bioavailability and half-life and a somewhat decreased degree of toxicity (Ginsberg, supra, 1996; Hirsch, supra, 1996; Sakuragawa and Takahashi,

Semin. Thromb. Hemost.

16 Suppl.:5-11 (1990); Pineo and Hull, supra, 1997; Boneu,

Semin. Thromb. Hemost.

22:209-212 (1996)). The ability of two kinds of clinical grade LMW heparins, FRAGMIN (Pharmacia-Upjohn; Kalamazoo Mich.) and LOVENOX (Rhone-Poulenc Rorer; Collegeville Pa.), to inhibit selectin binding was compared to unfractionated pharmaceutical heparin (FIG.

8

). The IC

50

values of the LMW heparins for L-selectin and P-selectin binding were at or higher than the recommended therapeutic levels of unfractionated heparin in ELISA inhibition experiments against immobilized SLe

x

and in HL-60 cell attachment inhibition experiments (Table 5). Thus, the two LMW heparins are much poorer inhibitors of L-selectin and P-selectin binding to sialylated ligands, including the naturally occurring PSGL-1 ligand, than unfractionated pharmaceutical heparin. The difference in binding inhibition between unfractionated and LMW heparins appears to be due to smaller sized heparin fragments having less affinity or avidity for the selectins (see FIG.

6

).

The results disclosed herein demonstrate that L-selectin and P-selectin binding to SLe

x

and to PSGL-1 of HL-60 cells is inhibited by unfractionated pharmaceutical heparin preparations at concentrations 12-fold to 50-fold lower than those recommended for effective anticoagulation in vivo. These results indicate that patients undergoing heparin anticoagulant therapy can experience clinically significant inhibition of L-selectin and P-selectin function. However, the current switch from unfractionated heparin to low molecular weight anticoagulant forms of heparin likely results in a significant loss of the selectin inhibitory effect due to the decreased ability of LMW heparins to inhibit L-selectin and P-selectin binding. This relative lack of affinity may be due to selectins favoring higher order carbohydrate structures. As disclosed herein, administration of low dose unfractionated heparin provides a treatment option for acute and chronic diseases involving P-selectin or L-selectin mediated binding, particularly administration of heparin in an amount that does not produce substantial anticoagulant activity or undesirable bleeding in a subject.

Although SLe

x

and related structures may be therapeutically useful in a variety of disease states (Albelda et al.,

FASEB J.

8:504-512 (1994); Lowe and Ward, supra, 1997; Weyrich et al.,

J. Clin. Invest.

91:2620-2629 (1993); Ridings et al.,

J. Appl. Physiol.

82:644-651 (1997); Bevilacqua et al.,

Annu. Rev. Med.

45:361-378 (1994); Skurk et al.,

Am. J. Physiol. Heart Circ. Physiol.

267:H2124-H2131 (1994); Lefer,

Ann. Thorac. Surg.

60:773-777 (1995); Seekamp and Ward,

Agents Actions

41:137-152 (1993)), they have a number of significant drawbacks. As disclosed herein, the affinity of pharmaceutical compositions of unfractionated heparin for L-selectin and P-selectin is higher than that of oligosaccharides such as SLe

x

(Table 3), LOVENOX and FRAGMIN (Table 5 and FIG.

8

). In particular, the IC

50

calculated for inhibition of HL-60 cell attachment to L-selectin using unfractionated heparin is approximately 10-fold less than the recommended levels for anticoagulation (Table 5). The IC

50

values for P-selectin are even lower, and are approximately 50-fold less than the recommended therapeutic level for anticoagulant activity. Thus, the recommended level of unfractionated heparin for anticoagulation can completely inhibit L-selectin and P-selectin mediated binding in an in vitro binding assay.

In addition to FRAGMIN and LOVENOX, other forms of LMW heparins used for anticoagulant therapy are commercially available. As disclosed herein, these other LMW heparins also can be useful for inhibiting selectin binding at concentrations less than those used for anticoagulant therapies. Such forms of LMW heparin formulated for clinical use in anticoagulant therapies, therefore, also can be used in a method of the invention.

Many reperfusion injury situations such as those occurring in stroke and myocardial ischemia, in which inhibition of PSGL-1 based P-selectin and L-selectin interactions are a potential target (Table 1), are routinely treated with anticoagulating amounts of heparin with the intention of preventing further thrombosis. As disclosed herein, a result of administering these amounts of heparin is that clinicians may have inadvertently inhibited selectin mediated binding. Consistent with this view, the best effects of thrombolytic therapy for acute myocardial infarction occurs when heparin is included in the treatment regimen. The results disclosed herein indicate that heparin can provide protection from reperfusion injury by inhibiting P-selectin and L-selectin mediated binding.

The results disclosed herein, along with the established record of heparin as a therapeutic agent, indicate that heparin can be useful for inhibiting L-selectin or P-selectin based interactions using amounts lower than those required for anticoagulant therapy. Thus, the invention provides a method of inhibiting L-selectin or P-selectin binding in a subject, by administering to the subject an amount of heparin that does not produce substantial anticoagulant activity or undesirable bleeding in the subject. Further provided are methods of treating an L-selectin or P-selectin related pathology by administering to a subject having the pathology an amount of heparin that does not produce substantial anticoagulant activity or undesirable bleeding in the subject.

Particular acute and chronic conditions, in which P-selectin or L-selectin have a pathophysiological role can be treated using a method of the invention (Table 1). For example, undesirable immune responses in which the homing or adhesion of leukocytes, neutrophils, macrophages, eosinophils or other immune cells mediated by the interaction of L-selectin with endothelial cell ligands, can be inhibited by administering heparin to the subject according to a method of the invention. Inhibition of neutrophil adherence, for example, can interrupt the cascade of damage initiated by free oxygen radical secretion and related activities that result in tissue damage and loss of myocardial contractile function present in myocardial infarction. Similarly, P-selectin mediated adhesion of cells such as neutrophils and platelets can be inhibited in a subject if this activity is undesirable. Thus, the severity of chronic immune disorders or acute inflammatory responses can be reduced using a method of the invention.

When administered to a subject, heparin is administered as a pharmaceutical composition. Such pharmaceutical compositions of heparin are commercially available and protocols for heparin administration are well known in the art. Such compositions and administration protocols can be conveniently employed in practicing the invention. One skilled in the art would know that the choice of the particular heparin pharmaceutical composition, depends, for example, on the route of administration and that a pharmaceutical composition of heparin can be administered to a subject by various routes, including, for example, parenterally, particularly intravenously. The heparin composition can be administered by intravenous or subcutaneous injection, and administration can be as a bolus or by continuous infusion. In addition, mucosally absorbable forms of heparin can be administered orally, rectally or by inhalation, provided the amount of heparin attained in the blood does not exceed a concentration of about 0.2-0.4 units/ml plasma and does not produce substantial anticoagulant activity or undesirable bleeding in the subject.

Depending on the commercial source, pharmaceutical preparations of heparin for injection are supplied at concentrations from 1000 units/ml to 20,000 units/ml (Eli Lilly, Indianapolis Ind.; Elkins-Sinn, Inc., Cherry Hill N.J.; Wyeth-Ayerst Laboratories, Philadelphia Pa.; Pharmacia-Upjohn, Kalamazoo Mich.). Thus, pharmaceutical heparin preparations that are about one-fifth to one-tenth as concentrated can be used for administration to a subject in order to inhibit L-selectin or P-selectin mediated binding in the subject. For example, heparin pharmaceutical compositions of about 250-950 units/ml or 50-250 units/ml, or less can be prepared and used in the methods of the invention. Thus, the invention provides pharmaceutical compositions comprising less than 1000 units heparin/ml. Dilutions of more concentrated commercially available pharmaceutical heparin to achieve these lower concentrations of heparin also are provided.

The following examples are intended to illustrate but not limit the present invention.

EXAMPLE I

Binding of Heparan Sulfate Chains to L-Selectin and P-Selectin

This example demonstrates that heparan sulfate (HS) chains from endothelial cells bind to L-selectin and P-selectin.

A. Endothelial Heparan Sulfate Ligands for L-Selectin also Bind to P-Selectin but not to E-Selectin:

Affinity columns containing the L-selectin, P-selectin and E-selectin chimeric proteins were used to determine whether HS ligands released from endothelial cell HSPGs that bind to L-selectin, also can bind other selectins. Recombinant selectin chimeras consisting of L-selectin-Rg, P-selectin-Rg and E-select-in-Rg were produced as previously described (Norgard et al.,

Proc. Natl. Acad. Sci. USA

90:1068-1072 (1993); Aruffo et al.,

Proc. Natl. Acad. Sci. USA

89:2292-2296 (1992); Nelson et al.,

J. Clin. Invest.

91:1157-1166 (1993)). Affinity columns were prepared by immobilizing 0.5 mg of each selectin-Rg on 0.5 ml of protein A-SEPHAROSE (PAS; Sigma, St. Louis Mo.).

CPAE cells, a calf pulmonary artery endothelial cell line (ATCC Accession No. CCL 209), were used at or before passage 23 as a source of HS chains and were grown to 80% confluence. Human umbilical vein endothelial cells (HUVECs; Clonetics, San Diego Calif.) were used within the first 3 passages and also provided a source of HS chains. CPAE and HUVEC cells were labeled with (

3

H)GlcNH

2

(60 Ci/mmol; American Radiolabeled Chemicals, St. Louis Mo.) and the free HS chains were released from the (

3

H)GlcNH

2

labeled cell proteoglycans and purified as described previously (Norgard-Sumnicht et al., supra, 1993; Norgard-Sumnicht and Varki, supra, 1995; Roux et al.,

J. Biol. Chem.

263:8879-8889 (1988)). Similar amounts of the labeled HS chains in 100 mM NaCl, 20 mM MOPS (pH 7.4), 1 mM CaCl

2

, 1 mM MgCl

2

buffer were applied to each column. After washing the column, bound ligand was eluted using the above binding buffer, except CaCl

2

and MgCl

2

were replaced with 5 mM EDTA.

Most of the HS ligand rebound to the L-selectin column and was eluted with 5 mM EDTA (FIG.

1

A). The HS ligand also bound to the P-selectin column, however, it eluted very poorly with 5 mM EDTA (

FIG. 1C

, inset). Subsequent elution with binding buffer in which CaCl

2

and MgCl

2

was replaced with 20 mM EDTA resulted in recovery of all of the HS ligand from the P-selectin column. In contrast, no binding to the E-selectin column was detected (FIG.

1

B), although the E-selectin column did bind other ligands from carcinoma cells in a calcium-dependent manner and, therefore, was functional. (

35

S)-labeled HS chains from CPAE cells and labeled HS chains from HUVEC cells provided similar binding profiles to the L-selectin and P-selectin columns.

These results demonstrate that HS ligands obtained from CPAE and the HUVEC cells bound the L-selectin and P-selectin columns, but not E-selectin column.

B. Comparison of Calcium Dependence of Heparan Sulfate Chain Binding to L-Selectin and P-Selectin:

The calcium dependence of the HS chain interaction with L-selectin and P-selectin was determined. Aliquots of HS chains that previously bound to an L-selectin column were reapplied either to the same column or to a P-selectin column. The columns were washed, eluted with either 5 mM EDTA for L-selectin or 20 mM EDTA for P-selectin, and the fractions collected and monitored for radioactivity (FIG.

2

).

Omission of calcium from the binding buffer did not change the results, although calcium (approximately 50 μM) present in the 2 mM magnesium containing buffer may support L-selectin binding. The binding of HS chains to L-selectin was abolished in Mg

++

/EGTA buffers, in which calcium is present in negligible amounts (effective Ca

++

concentration 0.774 nM, Mg

++

concentration 3 mM; FIG.

2

). The calcium dependence of the L-selectin interaction was confirmed by demonstrating that 0.5 mM EDTA efficiently eluted HS chains from the L-selectin column (FIG.

2

A). In contrast to L-selectin, binding of the HS chains to P-selectin occurred even in the magnesium/EGTA buffer (see

FIG. 2B

, indicating that HS binding to P-selectin did not require exogenously added calcium.

Since the binding of the HS chains to P-selectin occurred in the presence of either calcium or magnesium, the binding requirements for exogenously added cations was determined. An aliquot of the HS-GAG ligand that had previously bound to an L-selectin column was dialyzed against water, then was adjusted into buffer containing 100 mM NaCl, 20 mM MOPS (pH 7.4). The P-selectin column was prepared by washing with 20 mM EDTA, incubating in 20 mM EDTA overnight at 4° C., then extensively washing with the MOPS/NaCl buffer. The HS-GAG aliquot was applied to the P-selectin column, washed with the same buffer and eluted with 20 mM EDTA. The fractions were collected and monitored for radioactivity.

Binding to P-selectin occurred in the absence of exogenously added cations (FIG.

3

); however, the ligand was eluted with 20 mM EDTA. These results show that L-selectin binding is divalent cation dependent, whereas P-selectin binding does not require exogenously added divalent cations.

EXAMPLE II

Binding of Porcine Mucosal Heparin to L-Selectin and P-Selectin

This example demonstrates that porcine intestinal mucosal (mast cell-derived) heparin (PIM-heparin) binds to L-selectin and P-selectin.

A. PIM-Heparin Shows Similar but not Identical, Binding to the Selectins:

The binding of commercially available PIM-heparin (Sigma, St. Louis Mo.) to L-selectin and P-selectin was analyzed. Briefly, the large heparin chains in this preparation were radiolabeled by adding a 5-fold molar excess of NaB(3H)

4

(approximately 1.4 to 7 mCi) to about 20 to 120 mmoles of di- to tetradeca-saccharide heparin in 100 μl of 0.2 M sodium borate buffer, pH 10. The reaction proceeded for 4 hours at room temperature and 40 μl of 1 M NaBH

4

was added. After one hour, 40 μl of acetone was added to quench the reaction. The next day, the samples were treated with 150 μl of 1.0 M ammonium formate buffer and stored at −80° C. until use.

Thawed samples were desalted using Bio-Gel P-2 columns (Bio-Rad, Hercules Calif.) in 100 nM ammonium formate buffer, pH 5. The void volume (Vo) and included volume (Vi) were determined by loading 400 μl 0.1% blue dextran (detected by color) in 100 mM ammonium formate buffer, pH 5, plus 200 mM NaCl (detected with AgNO

3

). Samples were eluted with 100 nM ammonium formate buffer, pH 5, into 500 μl fractions containing the Vo(3H) cpm which were pooled, lyophilized, dissolved in H

2

O, and stored at −20° C. SEPHADEX G-15 chromatography was used to remove additional contaminants.

Selectin affinity columns having a ratio of 1.7 mg of selectin-Rg to 1 ml PAS were prepared and used for the PIM-heparin studies. Aliquots of

3

H-labeled PIM-Heparin were applied to L-selectin, P-selectin or E-selectin affinity columns equilibrated in a buffer consisting of 20 mM Hepes, 125 mM NaCl, 2 mM CaCl

2

, 2 mM MgCl

2

, pH 7.45 (Koenig et al., supra, 1997). The column was washed and the bound PIM-Heparin eluted with the same buffer containing 2 mM EDTA (divalent cation-dependent component) in place of 2 mM CaCl

2

and 2 mM MgCl

2

. The second elution was with 20 mM Hepes (pH 7.45), 110 mM NaCl, 20 mM EDTA (divalent cation-independent component, EDTA acting as a polycarboxylate) and the fractions were collected as described above.

Using the L-selectin affinity column, 10% of the added PIM-heparin ran through, 58% of the heparin that bound to the column was eluted with 2 mM EDTA and the remaining 32% was eluted with 20 mM EDTA (FIG.

4

B). In contrast, for the P-selectin column, 21% of the material ran through, very little was eluted with 2 mM EDTA, and the 79% that remained bound to the column required repeated washing with 20 mM EDTA for elution (FIG.

4

C). For the E-selectin column, 100% of the PIM-heparin ran through (FIG.

4

A).

These results demonstrate that the binding of PIM-heparin to the selectins is similar to that of the HS chains obtained from endothelial cells, except that a fraction of PIM-heparin remained bound to L-selectin after elution with 2 mM EDTA.

B. Calcium Dependence of Heparin Binding to L-Selectin and to P-Selectin:

The calcium dependence of PIM-heparin binding to the L-selectin and P-selectin columns was determined as described above for endothelial HS chains. Aliquots of

3

H-labeled PIM-Heparin were applied to an L-selectin and P-selectin affinity column in a magnesium/EGTA buffer having a residual free calcium concentration of 774 pM. The columns were washed, eluted and the fractions were collected as described above.

L-selectin and P-selectin binding by PIM-heparin was similar to the endothelial HS chains (FIG.

5

). For the L-selectin column, 100% ran through; for the P-selectin column, 55% ran through, 3% eluted with 2 mM EDTA, and 42% eluted with 20 mM EDTA. Thus, binding to L-selectin was abolished in Mg++/EGTA buffers, in which calcium was present in negligible amounts. The fraction bound to P-selectin was lower (45% vs. 79%) than that detected under calcium-replete conditions indicating a partial calcium dependency of the initial binding (compare

FIG. 5

with FIG.

4

). The fraction that did bind in the absence of calcium required 20 mM EDTA for elution (FIG.

5

B).

These results demonstrate that calcium assists the initial binding of some PIM-heparin fragments to P-selectin. However, once bound, calcium is not required to maintain the interaction of the heparin chains with P-selectin.

EXAMPLE III

Binding and Characterization of Size-Defined Heparin Fragments

This example shows that L-selectin and P-selectin exhibit differential preferences in their binding towards heparin fragments.

A. Binding of Size-Fractionated Heparin Fragments to the Selectin Columns:

The binding of sized fragments of heparin to L-selectin and P-selectin was analyzed. Size-fragmented mixtures of PIM-heparin were prepared as previously described (Pervin et al., supra, 1995), labeled by reduction with NaB(

3

H)

4

and purified as described above. Aliquots of the labeled PIM-heparin fragments were loaded onto the L-selectin and P-selectin affinity columns, washed, and the bound ligand eluted and fractions collected as described above. Again, L-selectin binding was eluted by 2 mM EDTA, whereas most of the binding to P-selectin required 20 mM EDTA for elution, although a small portion was eluted with 2 mM EDTA (see Table 4).

The results shown in FIG.

6

and Table 2 indicate that the binding of these (3H)-heparin fragments to L-selectin and P-selectin is size dependent; substantial binding of tetradecasaccharides was observed (FIG.

6

D). Under the conditions of affinity column chromatography used here, binding constants in the low μM range are expected to be required for detection of binding. Consistent with this prediction, the tetradecasaccharides have IC

50

values in the low μM range for inhibition of L-selectin and P-selectin binding to SLe

x

(Table 3). Thus, the tetradecasaccharide mixture has IC

50

values that are about 6-fold to 10-fold greater than those reported for SLe

x

itself (tetradecasaccharides: 82 μM and 54 μM for P-selectin and L-selectin, respectively; and SLe

x

: 520 μM and 600 μM for P-selectin and L-selectin, respectively). Studies with Mg

++

/EGTA buffers confirmed the calcium requirement for L-selectin binding, indicating that the divalent cation dependence of this interaction was similar to that of bulk heparin and of the endothelial HS chains.

The fractionated (

3

H)-tetradecasaccharide heparin fragments were reapplied to the L-selectin and P-selectin columns to determine the reproducibility of the above results. In addition, cross-binding studies were performed to identify differences in the tetradecasaccharides that bound to L-selectin and P-selectin. (

3

H)-tetradecasaccharides (approximately 500,000 cpm) were loaded onto L-selectin and P-selectin columns and the fractions collected as described above (only 0.5% of each fraction was monitored). Run-through and eluted fractions were desalted using a CENTRICON unit (Amicon, Beverly Mass.) and the isolated pools were stored in 250 μl H

2

O at 4° C. Recoveries ranged from 50-99%.

For L-selectin, the pools were unbound (Pool A), slightly retarded (Pool B), retarded (Pool C) or eluted with EDTA (Pool D). For rebinding experiments, the pools were reapplied (2500 cpm) to the same column. For cross-binding experiments, (

3

H)-tetradecasaccharides (2500 cpm) that bound to L-selectin were loaded onto the P-selectin column and the (3H)-tetradecasaccharides (2500 cpm) that bound to P-selectin were loaded onto the L-selectin column. The columns were washed, eluted and the fractions were collected as previously described.

Rebinding of the pooled tetradecasaccharide fractions to L-selectin and P-selectin indicated that the binding profiles were reproducible (FIG.

7

and Table 4). In addition, 100% of the tetradecasaccharides that bound to L-selectin bound to P-selectin; these tetradecasaccharides were eluted with 20 mM EDTA (Table 4). Only a subfraction of the tetradecasaccharides that bound P-selectin also bound to L-selectin: 22% of the 2 mM EDTA-eluted and 51% of the 20 mM EDTA eluted material. Of the fraction that did not bind to L-selectin, 12% rebound to P-selectin and eluted from the column only with 20 mM EDTA.

These findings indicate that, while L-selectin and P-selectin both exhibit some selectivity in recognizing heparin tetradecasaccharides, P-selectin has a more permissive range of recognition.

B. Analysis of the Structural Features of the Tetradecasaccharides that Determine Binding Behavior to P-Selectin and L-Selectin:

The structural differences that account for differential binding was investigated by determining the type of tetradecasaccharides that bound to L-selectin and P-selectin. Aliquots (2000 cpm) of the pooled fractions isolated from the L-selectin and P-selectin columns were subjected to cleavage either with specific heparin lyases (I, II and III) as previously described (Lohse and Linhardt, supra, 1992; Desai et al., supra, 1993; Jandik et al., supra, 1994; Linhardt, supra, 1996) or by nitrous acid treatment at pH 1.5 or pH 4.0 (Conrad, supra, 1996). These treatments result in the cleavage of heparin chains, the type of which depends upon specific structural features or modifications of the heparin molecule.

Heparin lyase I (0.1-0.05 units) was used to digest (

3

H)-tetradecasaccharide, and 0.05 units were used to digest each of the pools at 30° C. for 8.5 hours in sodium phosphate/NaCl buffer, pH 7.1. Heparin lyase II (0.1 units) was used to digest all samples for 8 hours at 35° C. in sodium phosphate, buffer, pH 7.1. Heparin lyase III (0.2 units) was used to digest all samples for 11 hours at 35° C. in sodium phosphate buffer, pH 7.6. All digestions went to completion and were stopped by boiling for 5 minutes; samples were stored at −20° C.

Nitrous acid (HONO) degradation was performed by adding 100 uL of either the pH 1.5 or pH 4.0 HONO reagent to tubes containing the (

3

H)-tetradecasaccharide or each pooled fraction. The reaction proceeded for 10 minutes at room temperature, then was quenched with either 15 μl of 1 M Na

2

CO

3

for the pH 4.0 reaction or 35 μl of 1 M Na

2

CO

3

for the pH 1.5 reaction to obtain a final pH of 7.5. pH controls for each reaction were used for every experiment and consisted of replacing the sodium nitrite with sodium acetate for the pH 4.0 reaction, and barium nitrite with barium acetate for the pH 1.5 reaction. Samples were stored at −20° C. until FPLC (forward phase liquid chromatography) analysis.

The size of the treated fractions were determined by FPLC using a SUPERDEX 75 HR 10/30 column (Pharmacia, Alameda Calif.). The (

3

H)-tetradecasaccharide starting mixture, and the L-selectin and P-selectin binding and nonbinding (

3

H)-tetradecasaccharide pools were analyzed in parallel. The column was run isocratically in the same buffer used for the selectin-Rg-PAS columns (20 mM Hepes (pH 7.45), 125 mM NaCl, 2 mM MgCl

2

, 2 mM CaCl

2

). An FPLC system (Pharmacia, P-LKB-Pump P-500; P-LKB-Controller LCC-500 Plus; LKB-Frac 100) was used to elute the column at 1.0 ml/minute flow rate and collect 0.25 minute fractions after 6 min from the beginning of the run to 21 min resulting in 60 fractions per run being collected. The fractions were collected in scintillation vials and the radioactivity determined. The elution profile of this column was highly reproducible from run to run, as determined by addition of blue dextran (void column) and PIM-heparin sized mixtures ranging from disaccharides to tetradecasaccharides, which were radiolabeled with NaB(

3

H)

4

as described above. 50 μl of 1% blue dextran was added to every sample prior to loading onto the SUPERDEX 75 FPLC column to confirm run to run reproducibility.

L-selectin binding fragments were sensitive to Heparin Lyase I and HONO pH 1.5 and, therefore, the fragments included the more heavily sulfated and epimerized regions. The sensitivity to HONO, pH 4.0, treatment indicates that the L-selectin binding heparin tetradecasaccharide chains are enriched in the small amounts of free amino groups present in the mixture. The P-selectin binding component includes this fraction, as well as a fraction sensitive to Heparin Lyase III digestion which indicates the presence of less heavily modified tetradecasaccharide chains.

EXAMPLE IV

Heparin Inhibits L-Selectin and P-Selectin Binding

This example shows that pharmaceutical formulations of heparin can inhibit L-selectin and P-selectin binding.

A. Heparin Inhibits L-Selectin and P-Selectin Binding to SLe

x

:

The inhibitory properties of clinically formulated pharmaceutical heparin preparations were studied using ELISA inhibition assays.

ELISA inhibition assays were performed as previously reported (O'Connell et al.,

Proc. Natl. Acad. Sci. USA

93:5883-5887 (1996); Koenig et al., supra, 1997). Sterile 96 well ELISA plates were coated with 200 ng of polyacrylamide-SLe

x

(Glycotech; Rockville Md.) by overnight incubation at 4° C. in 100 μl of 50 mM sodium carbonate/bicarbonate buffer, pH 9.5. Plates were blocked for at least 2 hours at 4° C. with 200 μl of 20 mM Hepes, 125 mM NaCl, 2 mM CaCl

2

, 2 mM MgCl

2

, 1% protease-free BSA, pH 7.45 (osmolarity 290 milliosmoles) per well. During the blocking step, the selectin-Rg chimeras were separately preincubated at 4° C. with a peroxidase-conjugated goat anti-human IgG secondary antibody (Jackson Immunoresearch Laboratories, Inc.; West Grove Pa.) in assay buffer for approximately 1 hour.

Final selectin-Rg concentration was 20 nM, and the secondary antibody dilution was 1:1000. Potential binding inhibitors were serially diluted in assay buffer at twice the final required concentration. The selectin-Rg/secondary antibody stock was aliquoted into tubes containing an equivalent volume of inhibitor solution; buffer, alone, for the positive control; or buffer with 10 mM Na

2

EDTA, pH 7.5, for the negative control (final concentration 5 mM EDTA).

These tubes were preincubated at 4° C. for 30 minutes, then added to ELISA plates, in duplicates, to a well volume of 100 μl. After 4 hours at 4° C., the plates were washed three times with 200 μl per well of assay buffer at 4° C., followed by development with 150 μl per well of OLD solution (0.002 mg o-phenylenediamine dyhydrochloride (OPD)/ml in 50 mM sodium citrate, 50 mM disodium phosphate buffer, 1 μl/ml 30% H

2

O

2

, pH 5.2) at room temperature. Each well was sequentially quenched with 40 μl of 4 M H

2

SO

4

after a fixed amount of time. The absorbance at 492 nM was determined using SOFTMAX software and a microplate reader (Molecular Devices Inc., Menlo Park Calif.). Prior to curve fitting, the absorbance values were converted into percentages for comparative purposes using the formula: ({(average of duplicates)−(negative control)}/{(positive control)−(negative control)})×100) using the SOFTMAX software.

Two separate lots of pharmaceutical unfractionated heparin significantly inhibited L-selectin and P-selectin, but not E-selectin binding to immobilized SLe

x

(FIG.

8

and Table 5). The concentrations required were less than the recommended therapeutic range for anticoagulation; IC

50

values of 0.07-0.08 units/ml and 0.01-0.02 units/ml towards SLe

x

-binding of L-selectin and P-selectin, respectively (Table 5). In addition, two different types of clinical grade LMW heparins, FRAGMIN and LOVENOX, gave IC

50

values of 0.7-2.0 units/ml and 0.8-1.5 units/ml for inhibition of L-selectin and 1.5-2.0 units/ml and 0.8-1.0 units/ml for inhibition of P-selectin binding, respectively, which were at or higher than the recommended therapeutic levels (see FIG.

8

and Table 5).

B. Heparin Inhibits L-Selectin and P-Selectin Binding to HL-60 Cells:

The inhibitory properties of pharmaceutical heparin samples also were studied using HL-60 cell attachment assays. HL-60, a human promyelocytic leukemia cell line (ATCC; CCL 240)), was grown in RPMI 1640 media supplemented with 20% FBS (1% Pen-Strep/0.2% gentamycin) and used at or before 8 passages. The binding inhibition studies were performed by labeling HL-60 cells with (

3

H)-thymidine (1 pCi/ml media) for two days. After labeling, the cells, at a density of 2×10

6

cells/ml or less, were collected by centrifugation, washed three times with 20 ml of media, resuspended and the number of cells (1×10

4

to 1×10

5

cells/well) and radioactivity were determined. (0.3-1.7 cpm/cell). 1 pmole per well of L-selectin-Rg and P-selectin-Rg immobilized on 24 well tissue culture plates in 250 μl of 50 mM sodium carbonate/bicarbonate buffer, pH 9.5, for 8 to 12 hours at 4° C. were blocked for 2 hours at 4° C. with 500 μl per well of 20 mM Hepes, 125 mM NaCl, 2 mM CaCl

2

, 2 mM MgCl

2

, and 1% protease-free BSA, pH 7.45.

Serial dilutions of unfractionated and LMW heparins were prepared in duplicate as described above and aliquots of (

3

H)—HL-60 cells added for a final volume of 250 μl per well. Positive control wells with (

3

H)—HL-60 cells alone and negative controls including 20 mM EDTA were analyzed in triplicate. After incubation for 3-4 hours at 4° C. with gentle rotation, the wells were washed 3 times with 500 μl cold assay buffer lacking BSA. Bound cells were solubilized at room temperature for 10 minutes in 1% Triton X-100 and the lysate monitored for radioactivity. IC

50

values are expressed as a percentage of the positive control and calculated as described above for the ELISA inhibition experiments (Table 5).

Unfractionated heparin inhibits the interaction of HL-60 with L-selectin and P-selectin (FIG.

9

). The IC

50

values calculated are 0.02-0.03 units/ml for L-selectin and 0.003-0.01 units/ml for P-selectin (Table 5). These values are approximately 12-fold and 50-fold lower than the recommended therapeutic range for heparin anticoagulant therapy. These results indicate that levels of heparin less than those used in anticoagulation therapy are effective at inhibiting L-selectin and P-selectin binding. The results also indicate that the two low molecular weight heparins (FRAGMIN and LOVENOX) are much poorer inhibitors of L-selectin and P-selectin binding to sialylated ligands, including the Hl-60 ligand PSGL-1 (FIG.

9

and Table 5).

Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention.

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Number	Name	Date	Kind
5464815	Chamow et al.	Nov 1995	A
5527785	Bevilaqoa et al.	Jun 1996	A
5777081	Michalski et al.	Jul 1998	A
6596705	Varki et al.	Jul 2003	B1

Number	Date	Country
5090748	Sep 1993	CA
WO 9202232	Jul 1991	WO
WO 9408595	Apr 1994	WO

	Number	Date	Country
Parent	09/246993	Feb 1999	US
Child	10/414969		US

Inhibition of L-selectin and P-selectin mediated binding using heparin

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