WO1998007432A1

WO1998007432A1 - Beta2 integrin cell adhesion molecule inhibitors

Info

Publication number: WO1998007432A1
Application number: PCT/US1997/014478
Authority: WO
Inventors: Mary Shirley Perez
Original assignee: American Home Products Corporation
Priority date: 1996-08-21
Filing date: 1997-08-18
Publication date: 1998-02-26
Also published as: ZA977471B; AR009282A1; AU4152297A

Abstract

This invention concerns compositions and methods utilizing a compound of formula (I) for reducing or controlling inflammation and treating pathological conditions mediated by intercellular adhesion. More particularly, the present invention concerns compositions and methods for blocking or modulating the function of the Beta2 Integrin family of cell adhesion molecules.

Description

BETA₂ INTEGRIN CELL ADHESION MOLECULE INHIBITORS

This invention concerns compositions and methods for reducing or controlling inflammation and treating pathological conditions mediated by intercellular adhesion. More particularly, the present invention concerns compositions and methods for blocking or modulating the function of the Beta2 Integrin family of cell adhesion molecules.

Background of the Invention

Stanley P. Owen and B.K. Bhuyan describe the isolation of the crystalline antibiotic 2H-Pyran-2-one,6-(l,2-epoxypropyl)-5,6-dihydro-5-hydroxy acetate, which they refer to as U- 13,933, in their article "Biological Properties of a New Antibiotic, U- 13,933", Antimicrobial Agents and Chemotherapy- 1965, copyright 1966, pp. 804-807.

A.D. Argoudelis and J.F. Zieserl described further structural specifications of the antibiotic U-13,933 in "The Structure of U-13,933, A New Antibiotic", Tetrahedron Letters No. 18, pp. 1969-1973, 1966.

U.S. Patent No. 3,909,362 (Jiu et al.) discloses and claims a process for the production of the antimicrobial agents 5,6-dihydro-5(S)-acetoxy-6(S)-( ,2'-trans- epoxypropyI)2H-pyran-2-one, 5,6-dihydro-5(R)-acetoxy-6(S)-(r,2'-trans-epoxy- propyl)2H-pyran-2-one, and 5,6-dihydro-5(S)-acetoxy-6(S)-(l',2'-trans-propenyl)2H- pyran-2-one comprising growing Aspergillus sp. NRRL 5769 or Aspergillus sp. NRRL 5770 in an aqueous nutrient medium containing sitosterol or sitostenone and isolating the compounds from the medium.

The three metabolites taught in the Jiu et al. patent, above, were further explained as showing antimicrobial activity against C. albicans, and against specific bacteria, fungi and a trichomonad by S. Mizuba et al. in "Three antimicrobial metabolites from Aspergillus caespitosus" ', Can. J. Microbiol., Vol. 21, 1975, pp.

1781-1787.

In their article "Total Synthesis of (+)-Asperlin", Tetrahedron:Assyπ_etry Vol. 1, No. 3. pp. 137-140, 1990, Subban Ramesh and Richard W. Franck describe a stereochemically unambiguous synthesis of (+)-asperlin, a crystalline antibiotic from Aspergillus nidulans, from L-rhamnose and cite the configuration of the antibiotic as 4S, 5S, 6S, 7R.

Brief Descrintion of the Invention

The present invention comprises compositions and methods for blocking or modulating the function of the Beta2 Integrin family of cell adhesion molecules in a mammal, preferably in a human, the compositions and methods utilizing the compound having the structure:

which is named 6,7-Anhydro-2,3,8-trideoxy-D-galacto-oct-2-enoic acid.delta.-lactone 4-acetate, also referred to as 5,6-dihydro-5(S)-acetoxy-6(S)-(l,2-trans-epoxypropyl)- 2H-pyran-2-on.

Detailed Descrintion of the Invention

The present invention includes pharmaceutical compositions and methods of administering to a mammal, preferably to a human, the compound of this invention to inhibit intercellular adhesion mediated by the β2 Integrin family of cell surface molecules. Through this inhibitory activity the pharmaceutical compositions and methods of the present invention are useful in treating or inhibiting inflammatory and other pathological responses associated with cell adhesion. Moreover, the methods of the present invention are useful in treating or inhibiting the pathological conditions where leukocytes and lymphocytes cause cellular or tissue damage. Through this inhibitory action, the present invention includes methods comprising administering to a mammal in need thereof a therapeutically effective amount of the compound of the present invention to treat the conditions including, but not limited to, asthma, stroke, reperfusion injury, trauma, transplantation rejection, and atherosclerosis. The methods of this invention also include the treatment of autoimmune diseases including, but not limited to arthritis, lupus, multiple sclerosis, Type I diabetes, psoriasis, ixiflammatory bowel disease, and other inflammatory diseases and conditions.

This invention also comprises pharmaceutical compositions utilizing the compound of this invention. The compound of the present invention may be administered in any manner sufficient to deliver a therapeutic dose, including orally, parenterally or topically. Oral formulations will likely be preferred for most chronic ailments, with parenteral administrations being particularly useful for acute maladies, such as trauma or stroke. Topical formulations may be more desirable for certain autoimmune problems, such as psoriasis. These compounds may be administered neat or with a pharmaceutical carrier to a mammal in need thereof. The pharmaceutical carrier may be solid or liquid.

A solid carrier can include one or more substances which may also act as excipients, flavoring agents, lubricants, solubilizers, suspending or stabilizing agents, fillers, glidants, compression aids, binders or tablet-disintegrating agents; it can also be an encapsulating material. In powders, the carrier is a finely divided solid which is in admixture with the finely divided active ingredient. In tablets, the active ingredient is mixed with a carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain up to 99% of the active ingredient. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars such as sucrose, glucose, fructose and confectioner's sugar, lactose, dextrin, dry starch (e.g. corn, potato or tapioca starch), gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.

Liquid carriers are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The active ingredients can be dissolved or suspended in a pharmaceutically acceptable liquid caπier such as water, an organic solvent, a mixture of both, or pharmaceutically acceptable oils or fats. The liquid caπier can contain other suitable pharmaceutical additives such as solubilizers, emulsifϊers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid carriers for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably a sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil). For parenteral administration, the carrier can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid carriers are useful in sterile liquid form compositions for parenteral administration. The liquid carrier for pressurized compositions can be halogenated hydrocarbon or other pharmaceutically acceptable propellant.

Liquid pharmaceutical compositions which are sterile solutions or suspensions can be utilized by, for example, intramuscular, intraperitoneal or subcutaneous injection. Sterile solutions can also be administered intravenously. The compound can also be administered orally either in liquid or solid composition form.

Preferably, the pharmaceutical composition is in unit dosage form, e.g. as tablets or capsules. In such form, the composition is subdivided in unit dose containing appropriate quantities of the active ingredient; the unit dosage forms can be packaged compositions, for example, packeted powders, vials, ampoules, prefilled syringes or sachets containing liquids. The unit dosage form can be, for example, a capsule or tablet itself, or it can be the appropriate number of any such compositions in package form. The dosage to be used in the treatment must be subjectively determined by the attending physician.

The dosage requirements will vary with the particular pharmaceutical composition employed, the route of administration, the severity of the symptoms presented and the particular subject being treated. Projected daily dosages of active compound would be from about O.lμg/kg to about 100 mg/kg, preferably between 0.001-25 mg/kg, and more preferably between 0.01-5 mg kg. Treatment will generally be initiated with small dosages less than the optimum dose of the compound. Thereafter the dosage is increased until the optimum effect under the circumstances is reached; precise dosages for oral, parenteral, nasal, or intrabronchial administration will be determined by the administering physician based upon experience with the individual subject treated. Preferably, the pharmaceutical composition is in unit dosage form, e.g. as tablets or capsules. In such form, the composition is sub-divided in unit dose(s) containing appropriate quantities of the active ingredient; the unit dosage forms can be packaged compositions, for example, packeted powders, vials, ampoules, prefilled syringes or sachets containing liquids. The unit dosage form can be, for example, a capsule or tablet itself, or it can be the appropriate number of any such compositions in package form.

In addition, the compounds of this invention may be employed as a solution, cream, or lotion by formulation with pharmaceutically acceptable vehicles containing 0.5-5 percent, preferably about 2%, of active compound which may be administered to an affected area, such a surface area exhibiting the effects of psoriasis.

The following examples demonstrate the ability of the compounds of this invention to selectively inhibit, block or modulating the function of the Beta2 Integrin family of cell adhesion molecules. The specific compound referred to as asperlin in the Examples below is 6,7-Anhydro-2,3,8-trideoxy-D-galacto-oct-2-enoic acid.delta.- lactone 4-acetate, also referred to herein as 5,6-dihydro-5(S)-acetoxy-6(S)-(l,2-trans- epoxypropyl)-2H-pyran-2-one.

EXAMPLE I Asperlin inhibits the adhesion of activated. β2 integrin-expressing HL60 cells to recombinant soluble ICAM-1

Materials

HL60 cells were provided by the American Type Culture Collection (ATCC No. CCL240) and were used between passages 20 to 30 as the control β2 integrin expressing cells, following stimulation for exactly four days in culture media containing dimethylsulfoxide (DMSO). Culture media was comprised of RPMI 1640 (Gibco No. 3201870AJ) supplemented with Penicillin (100 Units/ml)/ Streptomycin Sulfate (100 mg/ml) (Gibco No. 600-5145AE), L-Glutamine (2mM) (Gibco No. 320-5030PG), and 10% heat inactivated fetal bovine serum (FBS, Hyclone No. A-1111L). Fetal bovine serum was heat inactivated by incubating in a 56°C water bath for 30 minutes. Stimulation of the expression of the β2 integrin on HL60 cells was achieved by growing the cells at a density of 2.5 X 10^ cells per ml of culture media for four days in the presence of 1.25% DMSO. Following the removal of the DMSO containing media, the β2 integrin on these cells was activated to a high affinity ICAM-1 binding state by the addition of 0.1 nM of (PMA) and PMN Buffer. This buffer was comprised of Hank's balanced salt solution (HBSS) supplemented with 1.2mM calcium chloride, ImM magnesium chloride, 2% glucose, 20μM HEPES buffer. The ICAM-1 used to measure the adhesion of β2 integrin-bearing cells was obtained from cloning and expression of recombinant, soluble, human ICAM-1 using a baculovirus expression system and standard molecular biology technology. Soluble ICAM-1 was cloned by cleaving ICAM-1 DNA, purchased from R & D Systems, with restriction enzymes to obtain DNA that codes for the soluble (non-membrane) form of the protein. This DNA was then cloned into a baculovirus vector and the expression of soluble ICAM-1 was achieved in Sf9 cells using a kit obtained from Invitrogen Corporation. The soluble ICAM-1 was purified by passing the media from the ICAM-1 expressing cells over an anti-ICAM-1 antibody-linked sepharose column prepared using a standard immunoaffinity chromatography kit obtained from Pierce Inc.

Procedure

200ng of purified, recombinant, soluble ICAM-1 contained in lOOμl of PBS was added to wells of a flat bottom, 96 well EIA/RIA plate (Corning No. 25801), covered with an adhesive backed plate sealer (Linbro No. 76-401-05) and incubated for at least 18 hours at 4°C to allow ICAM-1 to bind to the assay wells, prior to the addition of the β2 integrin expressing HL60 cells.

HL60 cells were grown in 1.25% DMSO for 4 days and pelleted by centrifugation in a Sorvall RT6000 tabletop centrifuge for 5 minutes, 1000 rotations per minute (rpm), at room temperature. The resulting HL60 cell pellet was resuspended in 50ml sterile Dulbecco's phosphate buffered saline (dPBS) (Gibco No. 310-4040AJ). The resulting cell pellet was resuspended in 50 ml dPBS and the cell concentration was determined using a hemacytometer. The cells were pelleted as described above and resuspended in PMN Buffer to 15 to 20 X 10⁶ cells/ ml. The resuspended HL60 cells were fluorescently labeled by mixing the cells with an equal volume of a 25μM solution of Calcein AM (Molecular Probes No. C-1430) dye that had been dissolved in PMN Buffer. HL60 cells in the calcein AM solution was incubated in a 37°C waterbath for 10 minutes with intermittent swirling of the reaction tube. The labeling reaction was stopped by the addition of 13 ml of ice-cold PMN Buffer and the cells were pelleted by centrifugation at 2000 rpm for 5 minutes at 4°C.

The labeled cell pellet was resuspended in 15ml of ice-cold PMN Buffer and the cell density was determined using a hemacytometer. Cells were then pelleted by centrifugation at 2000 rpm for 5 minutes followed by resuspension in ice-cold PMN

Buffer to 2 XI 0^ cells/ 40μl. The labeled cell suspension was placed in the dark at room temperature while asperlin containing solutions were prepared.

Asperlin was solubilized in 100% DMSO at a concentration of 50mg/ml. This stock was then diluted to 400μg/ml with PMN Buffer and serial dilutions of the 400μg/ml asperlin solution were prepared using PMN buffer containing 2% DMSO to obtain solutions ranging from 200μg /ml to 1.56μg/ml.

The ICAM-1 assay plate was removed from 4°C and allowed to warm to room temperature and non-adherent ICAM-1 was aspirated from the assay plate using a multichannel pipettor. 200μl of 1% Tween-20 in dPBS was added to the ICAM-1 coated assay wells and the plate was incubated for exactly 2 minutes at room temperature. The 1% Tween-20/dPBS solution was removed from the wells by inverting the assay plate and shaking out the liquid. Wells of the assay plate were washed 4X with 200μl/well of PMN Buffer. After each wash, the plate was inverted and blotted on paper toweling to remove excess liquid. 50μl of the serially diluted asperlin was added to wells of the assay plate. The control wells received 50μl of PMN buffer containing 2% DMSO and the assay plate was incubated at room temperature for 10 minutes.

Calcein labeled cells, that had been gently mixed by swirling, were added to the wells in 40μl volumes equal to 2 X 10^ cells/ well, followed immediately by addition of lOμl of lxlO'^M PMA solution to all assay wells using a multichannel pipettor. The well contents were mixed using the same multichannel pipettor and the assay plate was incubated for 30 minutes in a 37°C, 5% CO2, humidified incubator.

The assay plate was removed from the 37°C incubator and total fluorescence of the labeled β2 integrin expressing cells in each well of the assay plate was measured using a fluorescent microtiter plate reader (Cytoflour, Millipore Corp). Non-adherent cells were aspirated from the wells of the assay plate using a multichannel pipettor. Wells of the assay plate were washed 3X with 200μl PMN buffer/ well. lOOμl of PMN buffer was added to wells of the assay plate. The fluorescence of the adherent, activated, β2 integrin expressing cells was measured using the fluorescent plate reader, as just described.

The percentage of B2 integrin expressing cells adhering to ICAM-1 wells was quantitated by the following equation: % Cells Bound = (Bound Cell Fluorescence -*- Total Cell Fluorescence) X 100

The percentage of inhibition of B2 integrin expressing cells adhering to ICAM-1 in the presence of asperlin was quantitated by the equation: % Inhibition of Cell Adhesion = 100% - [(Average % Cells Bound in the presence of asperlin ÷ Average % Cells Bound in control wells) X lOO]

The results obtained in this experiment, shown in the following table (Table I), demonstrated that asperlin blocks the adhesion of activated B2 integrin expressing cells to ICAM-1. The control wells, that do not contain asperlin, represent 0% inhibition in binding of B2 integrin expressing cells to ICAM-1. This suggests that asperlin is an inhibitor of β2 integrin mediated adhesion to ICAM-1.

EXAMPLE Π

Asperlin does not inhibit the adhesion of activated. βj_ Integrin -expressing U937 cells to human fifrpπggtin

Materials

U937 cells, a human monocyte-like, histiocytic lymphoma cell line, was acquired from American Type Culture Collection (ATCC No. CRL-1593). The cells were grown in the culture media described in Example I, materials. Cells were subcultured when the cell density was approximately 1x10*5 _ce]j_s p^- _mj j^e Bi integrins on U937 cells were activated to bind to fibronectin by the addition of PMN Buffer, described in Example I. The human fibronectin (Gibco, No. XOO1) was diluted to 3.5μg/ml with dPBS. A 1% Bovine Serum Albumin (BSA; Fraction V, ICN Corp., No. 810032) solution was prepared in dPBS and was sterile filtered using a 0.22mM disposable filter apparatus (Corning No. 25932-200) before use. The 1% BSA solution was used for blocking non-specific binding sites on the plastic wells of the assay plate. The assay plates used were Coming 96- well ELA RIA plates (No. 25801).

Procedure

350ng of human fibronectin contained in lOOμl of dPBS was added to wells of the assay plate. The control wells were filled with dPBS only. The assay plate was incubated at room temperature for exactly two hours to allow the fibronectin to bind to the assay wells. The fibronectin solution was aspirated from the wells of the assay plate and the wells were washed 3X with 200μV well of dPBS. The assay wells were filled with the 1% BSA solution. Additional wells, not coated with fibronectin, were also filled with the 1% BSA solution; these wells were used to measure non-specific cell adherence to the plastic assay plate. The assay plate was incubated for 30 minutes at room temperature. The 1% BSA solution was removed from the wells of the assay plate by aspiration using a multichannel pipettor. The assay wells were washed 3X with 200μl/well of dPBS. Approximately 50μl of dPBS was added to the assay wells to ensure that they would not dry out prior to the initiation of the assay.

U937 cells were harvested from culture, washed and labeled with calcein AM fluorescent dye as outlined in Example I, except that the concentration of calcein used to label U937 cells was 12.5μM and cells were sedimented by centrifugation at lOOOrpm for 5 minutes. After the final wash (see Example I), the U937 cells were resuspended in ice-cold PMN Buffer to 1.25 X 10^ cells/ 40μl. The cell suspension was placed in the dark at room temperature until needed. The asperlin solutions were prepared as described in Example I.

The 50μl of dPBS remaining in the wells of the assay plate was removed by inverting the assay plate and tapping on paper toweling. 50μl of the serially diluted asperlin solution was added to wells of the assay plate. Control wells received PMN buffer containing 2% DMSO and the assay plate was incubated for 10 minutes at room temperature.

Calcein labeled cells, that had been gently mixed by swirling, were added to the wells in 40μl volumes equal to 1.25 X 10^ cells/well and lOμl of PMA solution was immediately added to the wells of the assay plate and the assay was continued as described in Example 1. lOOμl of PMN buffer was added to all wells of the assay plate. The fluorescence of the adherent, activated Bi integrin expressing cells was measured using a fluorescent plate reader, as described in Example I.

The percentage of Bi integrin expressing cells adhering to fibronectin coated wells was quantitated by the equation: % Cells Bound = ((Bound Cell Fluorescence - Average Bound Fluorescence of the Non-specific Binding Control) ^÷ Total Cell Fluorescence) xlOO.

The percentage of inhibition of β l integrin expressing cell adhesion to human fibronectin in the presence of asperlin was quantitated, as described in Example I. The results obtained in this experiment, shown in the following table (Table II), demonstrate that asperlin does not block the adhesion of Bi integrin expressing cells to fibronectin. TAB E Π

Asperlin Total CeU Bound Cell Percent Average % Inhibition μg/ml Fluorescence Fluorescence Cells Percent CeU (fluorescence (fluorescence Bound Bound Adhesion units/well) units/well)

200 3019 2222 61 67 3

3475 2736 68

3455 2846 71

100 3407 2466 61 69 0

3534 2854 70

3738 3194 75

50 2960 2391 68 73 -6

3257 2791 74

3635 3203 77

25 3276 2404 61 69 0

3485 2870 71

3455 2969 75

12.5 3045 2331 64 70 -1

3248 2683 71

3495 2985 74

6.25 3194 2292 60 62 10

3248 2344 60

3294 2529 65

3.125 3594 3132 76 75 -9

3686 3140 75

3446 2895 73 TABLE π (Continued)

EXAMFLE m Asperlin does not inhibit the adhesion of HL60 Cells to E-selectin

The following experiments demonstrate that asperlin does not affect the binding of HL- 60 cells to recombinant, soluble, human E-selectin (rsE-selectin).

Ma&∑ial-.

rsE-selectin was prepared and purified using standard molecular biology techniques. HL60 ceUs were obtained from American Type Culture CoUection (ATCC) and maintained in culture media, as described in Example I, except that ceUs could be used in the assay at any passage number. Other materials required for the assay are as described in Examples I and π.

Procedure

200ng of purified, recombinant, soluble E-selectin contained in lOOμl of dPBS was added to weUs of a flat bottom 96 well EIA/RIA plate. The control wells were fiUed with dPBS only and the assay plate was incubated at 4°C for at least 18 hours to allow the rsE-selectin to bind to assay wells. The rsE-selectin solution was removed from the wells of the assay plate, washed, and blocked with 1% BSA, as described in Example II.

HL60 cells were harvested from culture, washed and labeled with calcein AM as outlined in Example I, except that the concentration of calcein used to label the HL-60 ceUs was 12.5μM and cells were centrifuged at lOOOrpm for 5 minutes. After the final ceU wash (see Example I), the cells were resuspended in ice-cold PMN buffer to 1.0 X 10-5 cells / 40μl and the ceUs were placed in the dark at room temperature untU added to the assay plate.

The asperlin solutions were prepared as described in Example I. The dPBS remaining in the wells of the assay plate was removed by inverting the plate and tapping it on paper toweling and the assay was performed as described in Example II. lOOμl of

PMN buffer was added to all wells of the assay plate and the fluorescence of the adherent HL-60 cells binding to rsE-selectin was measured using a fluorescent plate reader, described in Example I. The percentage of HL60 cells binding to E-selectin and the percentage of inhibition of HL60 ceU adhesion to E-selectin in the presence of asperlin were also quantitated as described in Example II.

The results obtained in this experiment, shown in the following table (Table

D3), indicate that asperlin does not inhibit E-selectin mediated HL60 cell binding.

TABLE m

Asperlin Total CeU Bound Cell Percent Average % Inhibition μg/ml Fluorescence Fluorescence CeUs Percent (fluorescence (fluorescence Bound Bound units/well) units/well)

200 1720 1124 52 48 8

1478 887 44

100 1832 1412 64 62 -19

1457 1096 59

0, Control 1526 977 49 52

1544 1017 51

1474 953 50

1559 1118 57

1554 1105 56

1637 1084 52

Non-specific 1520 128

Binding 1375 89 1351 199 1650 225 1647 270 1760 479 EXAMPLE IV

MTT Cvtotoxicitv Assay

This experiment describes the affect of asperlin on ceUular respiration

(mitochondrial activity) by measuring the reduction of the tetrazoUum salt MTT to formazan crystals (Moseman, et al). This assay served as a control to ensure that the decrease in the fluorescence observed in Example I, Table I, was not the result of the fluorescent dye leaking from the ceUs due to disruption of the ceU membrane. To address this issue, the mitochondrial activity of β2 and βi integrin expressing ceUs in the presence of asperiin was quantitated using a commerciaUy available MTT cytotoxicity kit (Promega No. G4100).

Materials

HL60 and U937 ceUs were purchased from the American Type Culture CoUection (ATCC) and maintained in RPMI 1640 culture media containing 10% heat inactivated fetal bovine serum (refer to Example I, materials). HL60 ceUs were used between passages 20 and 30. HL60 ceUs were stimulated by a four day exposure to DMSO, as described in Example I, materials. Falcon round bottom tissue culture plates (No. 3077) were used for the experiment. AU remaining reagents were supplied in Promega's MTT cytoxicity kit (Catalog No. G4100).

Procedure

The assay procedure described in the Promega MTT kit was used with the following changes.

Asperlin was solubiUzed in 100% DMSO at a concentration of 50mg/ml. The 50mg/ml asperlin stock was diluted to 400μg/ml with culture media containing 5% fetal bovine serum (FBS). This solution was further diluted to 80 and 20μg/ml with culture media supplemented with 5% FBS plus 2% DMSO. 50μl of the 20 and 80μg/ml asperiin solutions were added to wells of a sterile, round bottom 96 well assay plate. Control weUs received 50μl of culture media containing 5% FBS plus 2% DMSO. Duplicate assay wells were prepared such that U937 and HL60 ceUs could be evaluated on the same plate. The ceU density of βi integrin expressing U937, and β2 integrin expressing HL60 ceU cultures were determined using a hemacytometer.

Cells were harvested by centrifuging for 5 minutes at 1000 rpm, room temperature, in a Sorva RT600 bench-top centrifuge. The HL60 ceU peUet was resuspended in fresh culture media at a concentration of 5x10^ cells ml. The U937 ceU peUet was resuspended in fresh culture media at a concentration of 2.5x10-5 ceUs/ml. 2.5x10^ β2 integrin expressing HL60 cells, contained in 50μl, were added to wells of the assay plate using a multichannel pipettor. In dupUcate assay wells, 1.25x10^ βi integrin expressing U937 cells, contained in 50μl, were added to wells of the assay plate using a multichannel pipettor. Assay plates were incubated for either 30 minutes, 60 minutes, or 4 hours in a 37°C, 5% CO2, humidified incubator.

At the appropriate time point, 15μl of MTT reagent, supplied in the Promega assay kit, was added to each weU using a multichannel pipettor. Assay plates were incubated in the 37°C incubator, as just described, for two hours. The MTT reduction reaction was quenched, and the visible formazan crystals were solubUized by adding exactly lOOμl of Promega's solubilization buffer to each well. Assay plates were incubated for at least 18 hours in the same 37°C incubator just described to solubilize the formazan reagent. Assay plates were removed from the incubator and allowed to cool to room temperature. The optical density (OD) of each well of the assay plate was determined using a microtiter plate reader (Flow Labs) set at a wavelength of 580nm with a correction wavelength of 630nm

Replicate sample ODs were averaged. The percentage of inhibition of ceUular respiration (mitochondrial activity) by asperlin was quantitated by the foUowing equation:

% Inhibition = (l-(sample OD ÷Average Control OD) ) X 100

The results obtained in this experiment, shown in the following table (Table V), show that asperlin is not cytotoxic to either β2 or βi integrin expressing cells and, the reduction in β2 integrin mediated ceU adhesion by asperlin is not the result of an effect of asperlin on HL60 cellular function. TABLEV

Asperlin CeU Time Optical Average % Inhibition μg/mL Type Point Density Optical MTT

Density Reduction

40 HL60 30 min. .650 .649 27 .646 .650

10 HL60 30 min. .819 .819 8 .823 .815

0, Control HL60 30 min. .942 .895 .889 .891 .897 .875 .873

40 U937 30 min. 1.362 1.386 11 1.352 1.391

10 U937 30 min. 1.410 1.532 0 1.601 1.584

0, Control U937 30 min. 1.525 1.534 1.551 1.509 1.484 1.550 1.582 EXAMPLE V Asperlin inhibits β? integrin/ ICAM-1 homotvpic binding

The following experiment describes the inhibition of homotypic (ceU:ceU) binding of β2 and ICAM-1 expressing ceUs by asperlin. 8866 cells, a human B-ceU line, were supplied by Athena Neurosciences (San Francisco, CA). These ceUs constitutively express both the β2 integrin referred to as LFA-1, and ICAM-1 on the cell surface. In culture, 8866 cells spontaneously bind, or clump together, as a result of β2 integrin binding to ICAM-1. CeU adhesion blocking monoclonal antibodies against LFA-1 have been shown to completely block the binding of β2 integrin to ICAM-1, in the interaction referred to as ceUxell homotypic binding (Rothlein, et al.).

Materials

8866 cells were maintained in RPMI 1640 culture media (refer to Example I) supplemented with lOμM HEPES buffer (Gibco No. 15630-080). Cells were subcultured when the ceU density was approximately 1x10^ cells/ml. The anti-LFA-1 monoclonal antibody IOT16 (AMAC), was included in each experiment as a positive control inhibitor of the homotypic binding of LFA-1 to ICAM-1.

Procedure

8866 ceUs were harvested by centrifuging 5 minutes, at 1000 rpm, room temperature, in a SorvaU RT6000 centrifuge. The ceU pellet was resuspended in culture media and ceUs were counted using a hemacytometer. Cells were re-sedimented by centrifuging 5 minutes, at 1000 rpm, room temperature, in a SorvaU RT6000 centrifuge. The ceU peUet was resuspended in culture media at a concentration of 2x10^ ceUs ml. 2x10-5 cells contained in lOOμl were transferred to each well of a flat bottom, 96 well, tissue culture plate (Falcon No. 3072).

Asperiin was solubilized in 100% DMSO at a concentration of 20mg/ml, lOmg/ml, 5mg/ml, and 2.5mg/ml. These stocks were diluted with culture media to a concentration of 400μg/ml, 200μg/ml, 50μg/ml, and 25μg/ml, respectively. Asperlin was added to wells of the assay plate in volumes of 50μl per assay well. 50μl of culture media containing 2% DMSO was added to control wells of the assay plate. The monoclonal antibody IOT16 was diluted to 20μg/ml using culture media containing 2% DMSO. 50μl of the 20μg ml antibody solution was added to positive inhibition control weUs on the assay plate. A 200ng/ml solution of PMA was prepared using culture media, and exactly 50μl of this solution was added to weUs of the assay plate and the assay plate was incubated in a 37°C, 5% CO2, humidified incubator for two hours. The progression of homotypic binding was monitored by microscopic observations after 1 and 2 hours of incubation.

Inhibition β2 integrin dependent homotypic binding to ICAM-1 by asperUn was photographically recorded using a Nikon 35mm camera attached to a Nikon Diaphot 300 inverted microscope (filter setting NCBII, objective 10X, and tight setting of photo). Visual inspection of the assay plate after 1 hour of incubation indicated that the monoclonal antibody IOT16 inhibited 8866 ceU homotypic binding 100% (cells were not touching or binding to each other). Asperlin was visually observed to also inhibit cell-ceU binding in a dose dependent manner. Visual inspection of the assay plate after 2 hours of incubation indicated that the monoclonal antibody IOT16 completely inhibited (100%) 8866 ceU homotypic binding. Asperlin inhibited 8866, β2 integrin mediated homotypic binding to ICAM-1 in a dose dependent manner. These results confirm the effects of asperlin as a blocker of β2 integrin-mediated homotypic (ceU:ceU) adhesion.

Claims

What is Claimed:

1. A method for treating in a mammal a pathological condition in which leukocytes and lymphocytes cause ceUular or tissue damage, the method comprising administering to a mammal in need thereof a therapeutic dosage of a compound of the formula:

2. The method of Claim 1 wherein the mammal is a human.

3. The method of Claim 1 wherein the pathological condition is atherosclerosis.

4. The method of Claim 1 wherein the pathological condition is transplantation rejection.

5. A method for treating inflammation in a mammal, the method comprising administering to a mammal in need thereof a therapeutic dosage of a compound of the formula:

6. A method for inhibiting in a mammal pathological responses associated with intercellular adhesion mediated by the β2 Integrin famUy of cell surface molecules, the method comprising administering to a mammal in need thereof a therapeutic dosage of a compound of the formula:

7) The method of Claim 6 in which the pathological response associated with interceUular adhesion are asthma, stroke, reperfusion injury, trauma, transplantation rejection, arthritis, lupus, multiple sclerosis, Type I diabetes, psoriasis, inflammatory bowel disease or atherosclerosis.

8) A pharmaceutical composition which comprises a compound of the structure:

OAc and a pharmaceutically acceptable carrier. 9) The use of a compound of the Formula

in the preparation of a medicament for the treatment of:

a) a pathological condition in which leukocytes and lymphocytes cause ceUular or tissue damage;

b) inflammation; or c) pathological responses associated with interceUular adhesion mediated by the β_j Integrin famUy of cell surface molecules.